But like a champion, Williams regained it with the snap of a finger and played an absolutely brilliant decider to win 6-3, 3-6, 7-6 (2).
It was the most significant match since Kvitova's comeback from the horrendous hand injuries she suffered when she was stabbed by an intruder at her home last December.
The Czech responded well, though, breaking the Williams serve again early in the second and saving break points against herself, often accompanied by loud screams of "Pojd'!".
But it's her name - not those of her siblings or parents - that will go on that plaque and in the history books.
Steen: Kvitova is on a magical run coming back from her horrific hand injury, and although Venus is playing great, the h2h is well in the Czech's advantage, as is the momentum.
"Tiebreakers, you have to play smart, but you have to be aggressive", Williams said afterward.
At 37 years and three months Williams beat her own record, which she had set at Wimbledon this summer, as the oldest player to make the semi-finals of a Grand Slam event since 1994, when Martina Navratilova was Wimbledon runner-up at the age of 37 years and eight months. "I mean, she is really playing very, very well on the big stages", said Kvitova.
"I'm getting there slowly", Kvitova said on the eve of the tournament when asked if things were "back to normal".
The pair completed half of what could be an American clean sweep of the semi-finals, with Coco Vandeweghe and Madison Keys playing their matches on Wednesday. As a matter of fact, US Open is the only Grand Slam where she is yet to reach at least the semis.
The 24-year-old was unable to walk after undergoing surgery in January but has hit a remarkable hot streak, making the last four at three successive tournaments. No male or female qualifier has ever reached a US Open semi-final.
"It would be fantastic to play him in the final", Carreno Busta said of facing countryman Nadal in the final.
Kvitova will play seven-time major champion Venus Williams of the United States on Tuesday with a semi-final berth at stake.
- Williams hit five aces and 21 winners in a match lasting two hours and 34 minutes.
American Sloane Stephens also reached the quarterfinals by prevailing 6-3, 3-6, 6-1 over 30th-seeded German Julia Goerges. She raced to 5-1, each woman double faulted, but Williams served it out, 7-6 (2) in real style.
Petra Kvitova took solace from the plentiful evidence that she able to battle with the best again despite bowing out of the U.S. Open after a quarterfinal loss to Venus Williams on Tuesday.