Pegula knocked out world number one and 2022 champion Iga Swiatek in a crushing one-sided quarter-final to reach the last four at a Slam for the first time.
Navarro, also into her first semi-final at a major, defeated defending champion Coco Gauff in the fourth round.
AFP Sport looks ahead to Thursday's semi-finals at Flushing Meadows:
Jessica Pegula v Karolina Muchova
After losing six Grand Slam quarter-finals, 30-year-old Pegula is into her maiden semi and has not dropped a set over five rounds.
On Wednesday, she knocked out top seed and four-time French Open champion Swiatek for the loss of just six games.
It was Pegula's fourth win on hard courts against the Pole - after her three other wins, she went on to claim the title.
"Finally, finally, I can say I'm a semi-finalist," said Pegula.
"To do it at prime time against the number one player in the world it's crazy, but I knew I could do it."
After winning the Toronto title and finishing runner-up in Cincinnati, Pegula has won 14 of her 15 matches on the US hard-court swing, including a three-set victory over Muchova in Cincinnati.
Muchova is in the semi-finals in New York for a second successive year.
Muchova made the French Open final in 2023, rising to a career-high eight in the world, before suffering a serious right wrist injury which required surgery.
She only returned in June this year.
"There were a few bad days, and then I always try to just regroup and focus on the step that I could do, what's the first step I can do towards recovery," she said.
"I had a really great team of doctors around me and my team who is with me all the time supporting me. We worked a lot, we always stick together."
Emma Navarro v Aryna Sabalenka
Navarro lost the first round in both of her previous US Open appearances, but now the 23-year-old New Yorker is into her maiden Slam semi-final having knocked out defending champion Coco Gauff along the way.
Being the daughter of a billionaire father, Navarro has faced inevitable questions over her upbringing which included a private education.
"I did have access to resources as a kid, but I don't want that to take away from the fact that I've worked really, really hard to be where I'm at," said Navarro.
"I've put a ton of hours in, and I started training twice a day and getting in the gym from the time I was eight, nine years old. It's been a ton of hard work and dedication."
Her progress at the Slams this year has been on an upward curve - third round in Melbourne, last 16 at Roland Garros, where she lost to Sabalenka, and quarter-finals at Wimbledon.
The Minsk-born but Florida-based Sabalenka will be playing in her fourth successive US Open semi-final and is favourite to reach the championship match again having lost to Gauff 12 months ago.
The double Australian Open champion has dropped serve just four times at this year's tournament, winning 42 of her 46 service games.
She impressively demolished Olympic champion Zheng Qinwen for the loss of just three games in the quarter-finals.
"I'm going to try to put so much pressure on her (Navarro) and probably try to not let her do her stuff," said Sabalenka.
Despite the weapons at her disposal, Sabalenka's fastest serve of the tournament of 189.9 km/h (118 mph) was matched by Navarro in the first round.
