The Swede, who lost to Lee Westwood in a four-hole play-off here last year, is back on the PGA Tour for a second time thanks to finishing 13th on the Nationwide Tour money list last year and went around TPC Southwind in a five-under-par 65 to be nine under for the tournament following his 65 in the first round.
At the end of Karlsson's round his nearest challenger was American Colt Knost, who was three shots adrift, with the rest of the field a further two strokes further back.
World No?2 Westwood endured a difficult day, highlighted by a double-bogey 6 at 12, as he carded an even-par 70 to leave him eight shots off the pace following his first round of 69.
Karlsson began the round a stroke behind overnight leader David Mathis, who had dropped back into a share of third place on four-under after 14 holes, and the Swede shot ahead with four birdies in a flawless first nine.
Mathis had garnered his lead with a five-under 65 in Thursday's opening round when he finished with six birdies and one late bogey. Karlsson, 41, who began his round on the back nine, began his return home with a birdie at the first and another at six had moved him four shots clear.
And while a first blemish at the par-3 eighth meant he could not maintain that advantage, the Swede had set the benchmark with American trio John Merrick, Kris Blanks and Kevin Kisner just walking out for their rounds at four under. Keegan Bradley and Fabian Gomez both headed into the second round with 67s. Bradley is among the few here who could play their way into the US Open by winning at the TPC Southwind for a second tour victory since the last Open as he won the Byron Nelson Championship two weeks ago.