Status made her WWF debut as a heel on the March 19, 2000 episode of Sunday Night Heat. She appeared on stage to scout Test and Prince Albert. The next night on Raw is War, Test and Albert joined forces as the tag team T & A and Stratus began her first role in the company as their valet. It was during her stint managing T & A that Stratus took her first major bump in the ring, by being driven through a table by the Dudley Boyz at Backlash, after she had been taunting Bubba Ray Dudley for several weeks. In June, she was on the receiving end of a stinkface from Rikishi on Raw.
She also began managing then-heel, Val Venis to win the WWE Intercontinental Championship, but their partnership ended at SummerSlam after Venis lost the title. Stratus made her in-ring debut on the June 22 episode of SmackDown!, winning a tag team match with T & A against the Hardy Boyz and Lita. A storyline feud between Stratus and Lita developed after the match with Stratus attacking Lita on episodes of Raw and SmackDown!. This led to an Indian Strap match between the two women on the July 24 episode of Raw, which Stratus won with help from Stephanie McMahon. She finished the year competing unsuccessfully for the WWF Women's Championship numerous times, and separating from Test and Albert when the team disbanded.
In early 2001, Stratus became involved in an angle with WWF Chairman Vince McMahon, during a time when Vince's wife Linda was kayfabe institutionalized following a demand Vince had made for a divorce during an episode of SmackDown!. Vince and Stratus' relationship increasingly angered the boss' daughter, then-heel, Stephanie McMahon. On the February 19 episode of Raw, Stephanie and Trish were scheduled for a match, but it quickly got cancelled due to Stephanie getting stunned by Steve Austin. At No Way Out, Stratus and Stephanie squared off, with Stephanie scoring the victory after a run-in by William Regal. In the midst of a tag team match that pitted Vince and Stratus against Regal and Stephanie the next night on Raw, Stratus was the victim of a set-up by Vince, Stephanie and Regal. Regal executed his finisher, the Regal Cutter, on Stratus and Stephanie then dumped slop over Stratus' body. Vince stood over Stratus and he told her she was a "toy" with which he had "grown tired of playing with". The angle continued the next week on Raw, with Vince forcing Stratus to strip down to her black bra and panties in the ring and bark like a dog. The storyline came to an end at WrestleMania X-Seven, when Stratus slapped Vince during his match against his son Shane, turning face in the process.
Following her first face turn, Stratus began wrestling part-time. She teamed up with Lita against then-heels, Stacy Keibler and Torrie Wilson at InVasion. After suffering an ankle injury in the summer, however, she was sidelined for the following three months. This interrupted not only her recent venture into wrestling, but also an on-screen romance with Jeff Hardy and budding storyline with Team Xtreme. As she rehabilitated, she kept herself visible by co-hosting Excess. After returning in autumn, Stratus appeared at Survivor Series where she won the WWF Women's Championship for the first time in a six-pack challenge. Stratus was next involved in a feud with Jazz over the Women's Championship, where she retained the championship at the Royal Rumble, but dropped the championship to Jazz two weeks later on the February 4, 2002 episode of Raw. Stratus then attempted to regain the title for several months, including competing in a triple threat match at WrestleMania X8 against Lita and Jazz in her hometown of Toronto, Ontario, Canada, but failed to win the match. While chasing after the Women's Championship, Stratus won the WWE Hardcore Championship on May 6, pinning Crash Holly after Bubba Ray Dudley hit him over the head with a trash can. She lost the title to Steven Richards soon afterward however, due to the stipulation that the belt was to be defended 24/7 as long as there was a referee present. One week later, she won the Women's Championship for the second time in a tag team match with Bubba Ray Dudley where she faced Richards and Jazz, where the Hardcore Championship was also on the line. Stratus landed a hard metal trash can shot to Jazz's head, which badly weakened her and allowed Stratus to win easily. During this time, Stratus began wrestling solely on the Raw brand after being drafted in the WWF Brand Extension.
Stratus' second reign as champion came to an end on June 23, when she was defeated at King of the Ring by Molly Holly. The two Divas continued their storyline feud for the next three months. After a failed attempt to win the title in July, Stratus won the championship for the third time at Unforgiven. While feuding with Holly, Stratus was also involved in an angle with new Diva Victoria, who held a storyline grudge against Stratus, claiming she was betrayed by Stratus when they both worked as fitness models. The two competed in several title matches, with Stratus retaining until Survivor Series, where Victoria won the title in a Hardcore match. On March 17, 2003, Victoria and Steven Richards defeated Jazz and Stratus in a tag team match when Jazz walked out on Stratus. After the match, Jeff Hardy saved Stratus from an attack by Victoria and Richards and then kissed her, resulting in Stratus becoming Hardy's on-screen girlfriend once more. The two would talk and kiss backstage, compete as an intergender tag team, and come to each other's aid when in danger during singles competition. At no point was their previous relationship acknowledged. The storyline was suddenly dropped when WWE released Hardy in April.
At WrestleMania XIX, Stratus ended her feud with Victoria by defeating her and Jazz to capture her fourth Women's Championship, only to lose the title to Jazz at the following pay-per-view, Backlash. The Raw after Backlash, Eric Bischoff ordered a No Disqualification match between him and Stratus. If Stratus won, she would get a championship rematch the next week; if Bischoff won, he would get to spend a night with her. Bischoff would go on to win the match, though the stipulation was nullified when Linda McMahon confronted him after the match.
In the following months, Stratus was placed into an alliance with Gail Kim. It was short-lived, however, as Kim turned on Stratus and teamed with Molly Holly, putting the women in a storyline feud. The duo defeated Stratus and several tag team partners until Stratus allied herself with a returning Lita. The team defeated Kim and Holly in several matches, including a match at Unforgiven. Stratus began an on-screen romance with Chris Jericho during the November 10 episode of Raw when she agreed to go on a date with him. Subsequently, they participated in an intergender tag team match as partners on December 1. After the match, Stratus overheard Jericho talking to then-heel, Christian, who was involved in an on-screen romance with Lita at the time, about who could sleep with their respective woman first. One week later, Stratus and Lita confronted the men about their actions, leading to a feud between the two men and women which resulted in a "Battle of the Sexes" tag team match at Armageddon, which the women lost. A rematch the next night ended in a no contest. Her relationship with Jericho continued into the next year with a new angle of Stratus developing feelings for Jericho. Christian would also briefly turn face once again, but only revealed to be a hoax as he would attack and defeat her in a match ordered by Eric Bischoff. This would start a feud between Christian and Jericho, who was defending Stratus. During their match at WrestleMania XX, however, Stratus turned heel by betraying Jericho and siding with Christian. Stratus claimed her reasons for siding with Christian were that he was a "real man", and Jericho was a "love sick puppy". The duo feuded with Jericho for several months and competed in a 2-on-1 Handicap match at Backlash. The team of Stratus and Christian were joined by "problem solver" Tyson Tomko the next night on Raw.
Stratus won the WWE Women's Championship for a fifth time at Bad Blood on June 13. She defended the title until she suffered a legitimate broken hand in July that caused her to be out of action for approximately a month. Upon her return, she continued to defend the title against numerous challengers before losing the championship to Lita on December 6, when both women wrestled in the main event of Raw for the championship. Stratus recaptured the title for the sixth time a month later at New Year's Revolution, after Lita suffered a legitimate knee injury during the match. Stratus was originally booked to lose the championship back to Lita at WrestleMania 21, but due to Lita's injury, she wasn't cleared to wrestle. A new angle was then developed between Stratus and 2004 Raw Diva Search winner Christy Hemme over jealousy of Hemme's Playboy magazine exposure, with Stratus attacking Hemme with a Chick Kick and spray painting the word "slut" across her back. Stratus was challenged by Hemme, who later revealed she was being trained by Lita, for a championship match at WrestleMania 21, where Stratus successfully retained her championship. Stratus would then demand a rematch against Hemme the next night on Raw; before the match started, Stratus hit Hemme again with the Chick Kick, and re-injured Lita's knee. The next week, after losing a tag team match, Stratus was chased around the arena by Lita's storyline husband, Kane, narrowly escaping. The week after, Stratus almost got chokeslammed again onto the stage, but Viscera saved her. Stratus would then form a short-lived alliance with Viscera, who was ordered to protect her.
In May 2005, Stratus was sidelined with the Women's Championship after suffering a herniated disc, with the storyline explanation that Viscera had injured her at Backlash after she insulted him for losing to Kane. This left the company without a Women's Champion for four months, as Stratus remained the champion during the time of her injury, thus ignoring the 30 day clause stating that a champion must defend their respective title at least once every 30 days. She returned to Raw on September 12, 2005 as a face by siding with Ashley Massaro against Vince's Devils (Candice Michelle, Victoria and Torrie Wilson). The feud also involved the debuting Mickie James, who introduced herself as Stratus' biggest fan. In November, during the Eddie Guerrero Tribute Show, Stratus took part in an inter-promotional Divas battle royal that was won by SmackDown! Diva Melina. The next week, MNM (Joey Mercury and Johnny Nitro) kidnapped Stratus for Melina, who challenged a tied up and tape gagged Stratus to a match for the Women's Championship. The two fought at Survivor Series, with Stratus defeating Melina. Stratus and Mickie James continued teaming together in late 2005, while James became increasingly obsessed with Stratus. On the December 26 episode of Raw, the storyline between Stratus and James developed more, when James initiated an intimate kiss with Stratus under a sprig of mistletoe, causing a surprised Stratus to flee the locker room.
The odd relationship between Stratus and James continued into 2006, with the two Divas competing against each other in a title match at New Year's Revolution, where Stratus emerged victorious. Despite the defeat, James continued to be enamored of Stratus which made her feel uncomfortable, and on March 6, Stratus confronted James, telling her that she needed space. The duo briefly reconciled on the March 18 Saturday Night's Main Event XXXII, teaming together to defeat Candice Michelle and Victoria; however, after the match, James turned on Stratus and attacked her. A match at WrestleMania 22 saw Stratus lose the Women's Championship to James, ending her 448-day reign. During a rematch at Backlash, Stratus suffered a legitimate dislocated shoulder after taking a bump to the outside of the ring. While she was rehabilitating for six weeks, she continued to appear on-screen.
Stratus returned to the ring on June 26, where she had a Women's Championship match with Mickie James. Stratus would go on to lose the match, thus finally ending their nine-month long storyline. On the same night, she started a romantic angle with Carlito after he saved her from a double team attack by Melina and Johnny Nitro. They competed as a team, and won a mixed tag team match against Melina and Nitro at Saturday Night's Main Event XXXIII on July 15. As a couple, Stratus and Carlito briefly feuded with WWE Champion Edge and Lita (who turned heel) after the pair interrupted Stratus' title match with Mickie James. The two couples competed in several tag team matches, including a six-person tag team match where Edge, Lita and Randy Orton defeated Stratus, Carlito and John Cena after Orton RKO'd Stratus and Lita followed up with the pin. Stratus' last match on Raw occurred on September 11, 2006, where she defeated former rival Mickie James. At Unforgiven on September 17, in her hometown of Toronto, Stratus won against Lita with fellow Canadian Bret Hart's signature submission maneuver, the Sharpshooter. Her victory earned her her seventh and final Women's Championship, the most in WWE history, and she retired as the champion. The title was vacated soon after.
Stratus and Lita made a special appearance on December 10, 2007 during Raw's 15th Anniversary special, attacking Jillian Hall. The following year, Stratus appeared on Raw in Toronto on May 5, in a backstage segment involving Ron Simmons and Trevor Murdoch.
Stratus wrestled her first match in over two years on the December 22, 2008 episode of Raw, when she and John Cena defeated Santino Marella and his former on-screen protégé Beth Phoenix in a mixed tag team match. On September 14, 2009, Stratus served as the guest hostess of Raw, and participated in a six-person tag team match, teaming with Montel Vontavious Porter and Mark Henry to defeat Phoenix, Chris Jericho, and Big Show.
Stratus made a surprise appearance at the 2011 Elimination Chamber pay-per-view to announce that she would be a trainer on the revival of WWE Tough Enough and stopped LayCool (Layla and Michelle McCool) from attacking Kelly Kelly. The following month, on March 14, she lost a match to Vickie Guerrero due to interference from LayCool and Dolph Ziggler. After the match, John Morrison and Raw guest star Nicole "Snooki" Polizzi came to her aid. At WrestleMania XXVII, Stratus, Snooki, and Morrison defeated the team of Ziggler and LayCool. The night after WrestleMania, on Raw, Stratus and Morrison defeated Guerrero and Ziggler. She also appeared on Raw on June 6 and SmackDown on September 16.
The following year, on July 23, 2012, she made a guest appearance on Raw's 1000th episode.
On the January 28, 2013, episode of Raw, Stratus was announced as a WWE Hall of Fame inductee as part of the 2013 class, Stratus chose Stephanie McMahon to induct her into the Hall of Fame in April. The following year, Stratus inducted Lita into the WWE Hall of Fame.
On January 22, 2018, during the Raw 25 Years anniversary episode, Stratus was among a group of women honored as some of the greatest female superstars in the twenty-five year history of the show, along with The Bella Twins, Maryse, Kelly Kelly, Lilian Garcia, Torrie Wilson, Michelle McCool, Terri Runnels, Maria Kanellis, and fellow Hall of Famer Jacqueline. Six days later, Stratus was a surprise entrant in the inaugural all-women's Royal Rumble match at the event. Stratus entered in the final spot, number 30, and scored three eliminations, over Nia Jax, Mickie James and Natalya. Stratus' interaction with James garnered a considerable crowd reaction. Stratus was among the final five women in the match, before being eliminated by Sasha Banks.
On August 18, 2018 it was announced by WWE that she would make her in ring return to the company to compete at the Evolution event against Alexa Bliss. On the August 27, 2018 episode of Raw, Stratus made a surprise appearance interrupting Elias and confronting him for talking down on her hometown, the two exchanged insults, culminating in Stratus slapping him. Later in the night, Stratus along with Raw Women's Champion Ronda Rousey and Natalya, were confronted Alexa Bliss, Mickie James, and Alicia Fox, and Stratus then served as support along with Ronda Rousey during a one on one match between Natalya and Alicia Fox. She was then seen backstage during the show having a segment with Ronda and Natalya as well as the returning Bella Twins, Nikki and Brie. On the October 8 episode of Raw, Stratus would cut a promo against her then opponent at WWE Evolution, Alexa Bliss. Bliss, along with Mickie James came to the ring to confront Stratus, and challenged her to a tag match at Evolution. Almost immediately, Stratus would announce that Lita (who was originally scheduled to face James at Evolution) would be her tag partner. At the Evolution event, Trish and Lita defeated Mickie James and Alicia Fox (replaced Bliss due to injury).
At the end of July 2019, Stratus returned to the WWE as a guest of Jerry Lawler's "The King's Court" in–ring segment. During the segment, Charlotte Flair interrupted the two and issued a challenge to Stratus to wrestle at SummerSlam, which Stratus accepted. At the event, Stratus was defeated and subsequently announced her official retirement.
On February 27, 2023, Stratus made her return on Raw, helping Lita and Becky Lynch defeat Damage Control (Dakota Kai and Iyo Sky) for the WWE Women's Tag Team Championships by taking out Damage Control's leader, Bayley, who tried interfering. The following week, she announced that she would be coming out of retirement to team with Lita and Lynch to face Damage Control (Bayley, Kai, and Sky) in a six-woman tag team match at WrestleMania 39. At the event, Trish, Lita, and Becky were victorious against Damage Control. On the April 10 episode of Raw, Stratus teamed with Lynch to defend the WWE Women's Tag Team Championship against Liv Morgan and Raquel Rodriguez on behalf of an injured Lita. Morgan pinned Stratus with a roll-up, with Stratus assaulting Lynch following the match, effectively turning heel for the first time since 2005. The following week on Raw, Stratus revealed herself as the mystery assailant who attacked Lita. She was frustrated by the two appearing as Lynch's sidekicks and a perceived lack of respect for her impact on the women's division. On April 27, WWE announced that Stratus would be eligible during the second night of the WWE Draft. Stratus was once again drafted to the Raw brand in the fourth round as their seventh pick, the thirteenth of that night overall.
On May 8, Stratus taunted Lynch who had not been seen since losing the WWE Women's Tag Team Championships, and insulted her infant daughter Roux. However, Lynch would then return and attack Stratus with a Manhandle Slam. The following week, Lynch vowed to stand up for her family, and challenged Stratus for a singles match at Night of Champions in Saudi Arabia. The match was made official during a contract signing on Raw the following week. At the event, Stratus defeated Lynch with the help of Zoey Stark, who hid under the ring and attacked Lynch during the match. Stratus and Stark continued their partnership, and on the June 19 episode of Raw, Stratus won the qualification match for the women's Money in the Bank ladder match against Raquel Rodriguez by disqualification after Lynch attacked her during the match. On July 1, Stratus competed at the namesake event in her first appearance in a ladder match against Becky Lynch, Zoey Stark, Bayley, Iyo Sky, and Zelina Vega. Stratus worked with Stark throughout the match to prevent Lynch from winning, including a failed attempt to restrain her with handcuffs. Stratus, Stark and Lynch were defeated when Iyo Sky claimed the briefcase and won the match.
Lynch eventually earned a rematch against Stratus by defeating Zoey Stark in singles action on Raw, which was scheduled by WWE Official Adam Pearce for a future episode occurring two weeks later. On August 15, Stratus and Lynch's match ended in a double count-out. Adam Pearce declared that the two would have a future rematch inside of a steel cage, which would be a first for Stratus, at Payback. Stratus, who was aided by Stark, ended up losing the match at the event. After the match, Stark turned on Stratus after Stratus slapped and berated Stark.