The exit of the Czech, who reached last year's quarter-finals, leaves only three of the top 10 women's players in the running, with defending champion and world number three Sloane Stephens the highest ranked player left in the draw.
Kvitova beat Sabalenka in three sets at the Miami Open earlier this year but the often error-prone 20-year-old Belarusian showed she had tightened up her game considerably since that encounter.
Sabalenka made 15 unforced errors to Kvitova's 35 and won 80 percent of her first serves as she wrapped up the victory in one hour and 25 minutes.
She overpowered the twice Wimbledon champion from the back of the court too, winning 49 points from the baseline compared to Kvitova's 27.
"I really like to play on the big stages," Sabalenka said courtside.
"I really like to feel the support of the people. I didn't expect anything, I just went on the court and tried for every point and now I'm so happy.
"I think I put more balls in (than I did in Miami). It's enough."
Kvitova's defeat capped a bad day for the top seeds at Flushing Meadows with fourth seed Angelique Kerber, sixth seed Caroline Garcia of France and 10th seeded Latvian Jelena Ostapenko all failing to progress.
The quartet's exit follows that of top seed Simona Halep in the first round and second seed Caroline Wozniacki in round two.
There is still plenty of bite left in draw, however, with six-times champion Serena Williams firing on all cylinders and set to take on Halep's vanquisher, unseeded Estonian Kaia Kanepi, in the last 16 on Sunday.
(Reporting by Simon Jennings, editing by Nick Mulvenney)
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