The American went bogey-free on Thursday, carding a five-under-par 65 for an early advantage in Texas.
Johnson had four birdies on the front nine, including two in a row at the eighth and ninth holes, before adding another late in the day to finish a shot ahead of a four-man group.
Hunter Mahan made a blistering start to proceedings, eagling his opening hole before following that up with a birdie, but he faltered badly to end the round tied for second at four under.
The American is joined by compatriots Harris English and Robert Streb, and New Zealand’s Tim Wilkinson.
Two shots further back were Jimmy Walker, Tim Clarke, Brice Garnett, Freddie Jacobson, Bo Van Pelt, Jason Dufner, Jordan Speith, Ken Duke, David Hearn and Wes Roach.
Mahan led for much of the afternoon at the Colonial, with four birdies and an eagle catapulting him atop the leaderboard.
But he could not maintain that momentum, as he holed two bogeys at the 12th and 15th before ending his round with a double-bogey.
That opened the door for Johnson, who hit four birdies in a steady front-nine display.
Johnson fired six consecutive pars and he stepped things up at the 16th, birdying for sole possession of the lead.
Meanwhile, it was a difficult day for new world number one Adam Scott, who ended the day six shots off the pace.
The Australian scored a one-over-par 71, which included four bogeys, two of those coming back-to-back at the 17th and 18th before the turn.
Defending champion Boo Weekley was tied for 64th alongside Scott, who overhauled Tiger Woods for the top ranking on Monday.
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