Top seed Ashleigh Barty said it was "heartbreaking" to retire injured midway through the second set of her French Open second-round match against Poland's Magda Linette on Thursday.
The Australian, who won the title on her last visit to Roland Garros in 2019, left the court for medical treatment after losing the first set 6-1.
She had struggled with a hip problem during her first-round win over Bernarda Pera on Tuesday and called it quits at 2-2 in the second set on Court Philippe Chatrier on Thursday.
Barty also retired injured from the Italian Open during her quarter-final against Coco Gauff last month.
"It's heartbreaking," admitted the 25-year-old, who said she suffered the "new" injury in practice last weekend.
"We have had such a brilliant clay-court season, and to kind of get a little bit unlucky with timing more than anything -- to have something kind of acute happen over the weekend and just kind of run out of time against the clock is disappointing.
"It won't take away the brilliant three months that we have had, as much as it hurts right now."
It is Barty's earliest exit from a Grand Slam tournament since a second-round loss at Roland Garros in 2018.
She missed the majority of last season, including her French Open title defence, due to the Covid-19 pandemic, but had been in strong form in 2021.
Barty has won three titles this year, including on clay in Stuttgart, and lost to Aryna Sabalenka in the Madrid Open final.
"We have had an exceptional last two, two-and-a-half, three months, a little bit of a setback, and what happened today and this week here in Paris won't take away from that," she said.
"It's disappointing to end like this. I've had my fair share of tears this week. It's all good. Everything happens for a reason.
"There will be a silver lining in this eventually. Once I find out what that is, it'll make me feel a little bit better."
Barty will now turn her attention to getting fit in time for Wimbledon, which starts on June 28.
"I hope so. I really do," she said on her chances of being ready for the grass-court Grand Slam, which was cancelled last year.
"We do all the right things now, we continue to do the right things. Give ourselves the best chance."
Unseeded Linette will face Tunisian 25th seed Ons Jabeur in the last 32.
Barty's withdrawal leaves the tournament already without the world's top three-ranked women's players.
World number two Naomi Osaka stunned the sport by pulling out following a media boycott, saying she has suffered "bouts of depression" for three years.
Third-ranked Simona Halep, the 2018 French Open winner, withdrew before the event with injury.
That leaves world number four Sabalenka as the top remaining player in the draw, despite the Belarusian never having reached a Grand Slam quarter-final.
The world's top three will not be the only notable absentees from the third round, with injury-plagued former US Open champion Bianca Andreescu having fallen at the first hurdle.
The draw has opened up for the likes of reigning champion Iga Swiatek and history-chasing Serena Williams.
The 39-year-old Williams, still looking to equal Margaret Court's all-time record of 24 Slam titles, is the second-highest seeded player left in her half of the draw behind Sabalenka.