FORT WORTH, Texas, May 25, 2012 (AFP) - Dufner's six-under par effort gave him a 36-hole total of 11-under 129.
He was two strokes ahead of overnight leader Zach Johnson, who followed up his opening 64 with a 67 for 131.
Americans Bo Van Pelt and Tommy Gainey were equal third on seven-under 133. Van Pelt also posted a 64 while Gainey carded a 67.
Dufner, fresh off a victory in the Byron Nelson Classic in nearby Irving last week, will be trying to join legendary Ben Hogan as the only players to win both USPGA Tour events in the Dallas-Fort Worth area in the same year.
In 1946, Hogan won the Colonial and the Dallas Invitational, which later became the Byron Nelson Classic.
Dufner would be the first player to win the tour's two North Texas tournaments in consecutive weeks.
He said he would "hopefully continue to play some good golf and maybe make a little bit of history."
Dufner broke through for his first US tour title in his 163rd start on April 29 with a playoff victory at the Zurich Classic in New Orleans.
He was married the following weekend, then joined Hunter Mahan as 2012's only repeat winners by taking the Byron Nelson crown.
He has led or shared the lead after 11 of his last 34 rounds.
On Friday, he teed off on 10 and birdied the par-three 13th and par-four 17th, before launching a run of three straight birdies at the par-five first. He grabbed one more birdie at the par-four fifth hole.
Johnson launched his round with an eagle at the par-five first, where he drained an 18-foot putt. He made a 12-footer at the second for the first of his four birdies, but also had three bogeys.
"I hit a couple of bumps along the way," said Johnson, whose round was still good enough to get him in the final group on Saturday.
"Anytime you play late on Saturday is a good feeling," he said.
Defending champion David Toms was among the notables missing the cut, which fell at 143.
US Ryder Cup captain Davis Love also missed the cut as did Japanese star Ryo Ishikawa, who followed up a first-round 74 with a 70 for 144.
Leading second-round scores on Friday in the $6.4 million PGA Tour Colonial (USA unless noted, par-70):
129 - Jason Dufner 65-64
131 - Zach Johnson 64-67
133 - Bo Van Pelt 69-64, Tommy Gainey 66-67
134 - Tom Gillis 65-69
135 - Ryan Palmer 68-67, Harris English 65-70
136 - Rickie Fowler 68-68, John Huh 70-66, Ryan Moore 67-69, Bryce Molder 72-64
137 - Kevin Chappell 70-67, Andres Romero (ARG) 66-71, Kyle Reifers 65-72, Charlie Wi (KOR) 68-69, Greg Owen (GBR) 69-68
138 - Louis Oosthuizen (RSA) 71-67, Bobby Gates 71-67, David Mathis 71-67, Jim Furyk 69-69
139 - Greg Chalmers (AUS) 70-69, Carl Pettersson (SWE) 70-69, Vijay Singh (FIJ) 70-69, Charley Hoffman 69-70, Tim Clark (RSA) 70-69, Seung-Yul Noh (KOR) 70-69, John Daly 70-69, Sergio Garcia (ESP) 66-73, Sunghoon Kang (KOR) 70-69