As temperatures topped 90 degrees Fahrenheit (32 Celsius), former champion Johnson rattled up four consecutive birdies in blustery conditions en route to a flawless six-under-par 64 at Colonial Country Club where he clinched the 2010 title.
"Today was great," Johnson, the 2007 Masters champion, said. "I struck the golf ball really nice, real solid, and I gave myself a lot of opportunities.
"I made some long putts but I missed some shorter ones. It probably evened out at the end. I just hit some quality shots. I've got zero complaints."
Dufner, winner of the Byron Nelson Championship on Sunday who is seeking a third US PGA Tour victory in just four weeks, opened with a 65 to finish level with fellow Americans Tom Gillis, Kyle Reifers and rookie Harris English.
Spaniard Sergio Garcia saved par from greenside bunkers on his last two holes for a 66 to share sixth place with Americans Chris DiMarco and Tommy Gainey, and Argentina's Andres Romero.
Johnson, who has not triumphed on the US PGA Tour since he landed his seventh career win at Colonial, set the tone for a memorable day with a 40-foot birdie putt on his opening hole, the par-four 10th.
He then sank putts from between 14 and 22 feet to birdie the 13th, 14th, 15th and 16th before reaching the turn in five under. Johnson picked up one more shot, at the par-four third, as winds gusted up to 30 mph (48 kph).
Dufner, who was among the day's late starters, launched his round with five birdies in his first eight holes before finishing just one stroke off Johnson's sizzling pace.
"It was a nice round," the laid-back American said after mixing seven birdies with two bogeys. "The winds were up a little bit this afternoon and that made it a little tougher to gauge distances and pick the right clubs.
"Obviously a couple of wins here in the last couple of events have boosted my confidence a little bit but I've felt really good about my game since about March.
"I'm on a good roll, playing a lot of good rounds, and I felt good coming into this week. I'm looking forward to the next three days here."
American world number six Hunter Mahan, a double winner on the 2012 US PGA Tour, opened with a 69 while fifth-ranked compatriot Matt Kuchar, who clinched the prestigious Players Championship two weeks ago, carded a 72.
American veteran David Toms, who claimed last year's Colonial Invitational by one shot over South Korean Charlie Wi, launched his title defence with a 74.