Kvitova then survived a two deuce game after serving an ace out wide to hold serve to reach 6-2, 5-1 after 59 minutes of play.
Serving to stay in the match, Williams went down 0-30 before striking a crosscourt forehand winner to reach 30 all.
Williams then held serve to reach 2-5 after Kvitova struck two groundstroke errors.
Serving for the match, Kvitova faced break point at 30-40 after Williams struck a forehand down the line winner.
The Czech then reached deuce twice on her serve before she sent a crosscourt backhand long to drop serve and reach 5-3.
Serving to stay in the match, Williams sent an ace down the tee to go ahead 15-0.
Kvitova netted a forehand down the line to put the American ahead 30-0.
Williams double faulted then netted a crosscourt forehand to reach 30 all.
A crosscourt forehand from Williams sent long put the American down match point.
Williams netted a backhand down the line to end the match after one hour and 13 minutes of play.
After winning the Australian Open and the Miami Open and withdrawing injured in the semifinals at Indian Wells, Williams had been undefeated this year and entered today’s match with a career head to head versus Kvitova of 5-0.
However today Kvitova’s mix of offense and consistency would prove too much for an erratic Williams to overcome on clay, her least favorite surface.
Kvitova, a powerful left hander currently ranked four, won Wimbledon in 2011 as well as last year in her only grand slam victories.
Prior to today, Williams had won 27 matches in a row and had not lost a WTA Tour match since October.
"I think she just played well today. She went for broke on every serve, every return,” Williams said before adding “I was very slow off the mark.”
“After my serve I wasn’t moving as well as I should have. You know, I wasn't in it.”
"I was exhausted. I wasn't Serena today. I think that was the main thing."