Tiger Woods struggled to a second consecutive even-par 70 that left him one shot from missing the cut and nine strokes behind the lead going into Saturday's third round.
Guthrie, 23, was flawless as he carded seven birdies at PGA National to move into the driver's seat with a one-stroke lead over compatriot Michael Thompson (65).
Canadian Graham DeLaet (68) and American Boo Weekley (67) were a further shot off the pace.
With McIlroy gone and Woods out of contention, there was an unfamilar look atop the leaderboard, and those concerned with ratings will hope some of the seasoned names can put themselves in the frame for Sunday's final round.
Britain's Lee Westwood (68) and Australian Geoff Ogilvy (66) put themselves in contention as they are part of a group of six players sitting three shots back of Guthrie.
Westwood said he was taking a cautious approach to the challenging course, which on Friday remained very soft with frequent mud on balls.
"It's a tough setup and it's one of the more demanding tests of the year," said the Englishman. "There's trouble out there; if you get a good game plan, then you can get around it. Once you start getting a bit cute and chasing, it can bite you."
Woods had his share of trouble, needing to get out of difficulty on the final two holes to make sure he made the cut.
On the 17th hole Woods got up and down despite a tricky bunker shot and managed par on the par-five 18th despite finding the fairway bunker on his tee shot and going way wide into the spectator area with his second shot.
"I didn't quite have my game like I did yesterday. I hit it much better yesterday, but I putted better today so it all evened out," said Woods.
"I've got to get something going here. There's 70-plus guys within nine shots of the lead, just pretty bunched. Anybody in this tournament can win it.
"So hopefully tomorrow I can get it going, at least give myself a chance going into Sunday."