British Open champion Francesco Molinari and former Masters winner Adam Scott were among those threatening the lead.
Three weeks after leading into the final round at the British Open, Kisner showed himself again as a man for the big occasion when he bagged four birdies to six holes in perfect morning conditions at Bellerive.
The diminutive 34-year-old from South Carolina vaulted to seven under par, one stroke ahead of overnight leader Gary Woodland.
World number 44 Woodland, whose career has been hindered by a mediocre short game, did not rest on his laurels after carding an opening six-under 64.
He remained as he started the day at six-under, while big guns Molinari, Zach Johnson and Brandon Stone were two shots off the pace.
Three weeks after a magnificent victory at the British Open, Molinari continued his good form with three birdies in the first seven holes.
But not all the early starters made hay, with world number one Dustin Johnson among those failing to take advantage of the warm and calm morning.
He was three under at the turn after one birdie and one bogey.
Johnson was outplayed by another member of his group, Australian Adam Scott, who used his textbook swing to notch four birdies in an outward half of just 31 strokes.
Scott, the 2013 Masters champion, hit a series of precise approach shots and wielded his long putter with unerring accuracy.
A short birdie at the 18th, his ninth, took him to within three of Kisner.
Japan's Satoshi Kodaira recorded the first eagle of the championship when he drove the green at the par-four 11th and sank a five-foot putt. He was one over for the tournament.
Tiger Woods, Rory McIlroy and defending champion Justin Thomas were among half the field with late tee times.
Huge galleries turned out early in an area starved of big-time golf, with a long traffic jam at the nearby freeway exit forming before dawn as fans tried to beat the rush, mostly without success.
(Reporting by Andrew Both; Editing by Christian Radnedge)
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