In the opposite end of the draw, new world No.1 Elena Rybakina will take on home favourite Coco Gauff in the other last-four clash.
For the first time since Wimbledon in 2009, all four top seeds at a grand slam have reached the semi-finals, with Sabalenka, Rybakina and Gauff all former major champions and third seed Pegula seeking a maiden grand slam title.
Sabalenka has reached the US Open semi-finals for a sixth consecutive year and is now two wins away from becoming the first woman since Serena Williams from 2012 to 2014 to win three straight titles in New York.
The Belarusian had not dropped a set before surviving her toughest test of the tournament on Tuesday, edging Wimbledon champion Linda Noskova 7-6 (7-1) 3-6 7-6 (10-7).
The 28-year-old will face a familiar opponent in third seed Pegula, with their rivalry returning to Flushing Meadows for a third straight year after Sabalenka denied the American the title in 2024 and then beat her again in last year's semi-finals.
Sabalenka leads their head-to-head 9-4 but Pegula has won two of their last three encounters, including a victory in the Berlin Open semi-finals earlier this year in which she took the deciding set 6-0.
"I think it's fun to look at the rivalry we've had. She's gotten me here the last couple of years," Pegula said. "I have been able to beat her a few times, maybe on a smaller stage, so I think that's going to be kind of a hurdle for me to get over."
Pegula advanced by fighting back to beat fellow American Emma Navarro 3-6 6-4 6-3, reaching her second major semi-final this year after the Australian Open.
The 32-year-old has also reached five finals this season, the most on the WTA Tour.
Sabalenka will enter Thursday's semi-final after surrendering top spot in the world rankings, which Rybakina secured with her quarter-final victory over Olympic champion Zheng Qinwen on Wednesday.
The Australian Open winner will become the 30th woman to top the WTA rankings and the first from Kazakhstan to reach world No.1 when the new rankings are released after the tournament.
Sabalenka, who had held the top spot since October 2024, said she would gladly trade it for another US Open title.
"I'll be more than happy," she said. "Yes, three-peat. Sounds great."
Rybakina's reward for reaching the semi-finals for the first time in New York is a meeting with fan-favourite Gauff, who produced a stunning comeback to beat fifth seed and fellow French Open winner Mirra Andreeva 2-6 7-6 (9-7) 6-2.
The 2023 US Open champion was on the brink of defeat in the second set before saving two match points and turning the match around.
Gauff's victory put her into the semi-finals at a second straight grand slam after her run to the last four at Wimbledon, and she will have the home crowd behind her as she bids to clinch a second US Open title.
Rybakina will bid for a third grand slam title, while Gauff will have revenge on her mind after the Kazakh beat her in three sets in last month's Canadian Open semi-finals.
The pair are tied 1-1 in their previous meetings and will face off for a place in Saturday's final.
US OPEN ORDER OF PLAY ON THURSDAY (prefix number denotes seeding):
ARTHUR ASHE STADIUM
1-Aryna Sabalenka (Belarus) v 3-Jessica Pegula (US)
4-Coco Gauff (US) v 2-Elena Rybakina (Kazakhstan)
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