Dufner fired a 6-under 64 on a hot, windy day and, for the fifth time this year, is out front heading into the weekend of a PGA Tour event.
His 36-hole total of 11-under 129 at the Colonial Country Club was two shots better than 2010 champion Zach Johnson, four shots better than Bo Van Pelt and Tommy Gainey and five shots better than everybody else.
Dufner is looking for his third victory in four starts. He won in New Orleans April 29, got married the following week, honeymooned at the Players Championship (where he finished tied for 68th), and then won last Sunday at the Byron Nelson Championship by sinking a 25-foot birdie putt on the final hole.
A victory this week would make him the first player since Ben Hogan 66 years ago to win both the Dallas and Fort Worth stops on the tour in the same season.
Dufner began his round on the back nine and made the turn in 2-under 33. He birdied four of the first five holes on the front side, narrowly missing an eagle putt on the first and chipping in for his final birdie of the day at the difficult, par-4 fifth.
He has broken 70 in 11 of his last 17 competitive rounds.
Johnson had the lead at the end of the first day, but when he teed off Friday he was five shots behind Dufner. Johnson eagled the opening hole, but suffered three bogeys in a round of 67.
Van Pelt equaled Dufner's 64 as the low round of the tournament and Gainey shot a 67.
Players Championship winner Matt Kuchar was at 141.
John Daly completed 36 holes at 139 and made the cut in a PGA Tour event for the third time this year. His best finish in the United States during 2012 has been a tie for 51st at the Transitions Championship.