Mathis completed 36 holes at 14-under 130, which was one shot off of Stan Utley's 2002 tournament record.
Play rotates over three courses for the first three rounds with the professionals playing with amateur partners.
The courses are the par-71 Thornblade Club, the par-72 Bright's Creek Golf Club and the par-72 Carolina Country Club. Thornblade will host the final round.
Mathis posted his round on Friday at the Bright's Creek Golf Club.
Matt Weibring also shot a 65 in the second round at Carolina Country Club and is alone in second place at minus-12.
Matt Hendrix carded a four-under 68 at Bright's Creek and is third at 11-under 133. Greg Chalmers posted a 67 at Bright's Creek and is fourth at 10-under 134.
Mathis parred his first two holes, then jump started his round with a great 4-iron. At the par-3 third, Mathis hit a 4-iron to a foot and tapped in for birdie.
He made it two in a row with a six-footer at the par-4 fourth. Mathis took advantage of the next par three, the sixth. He rolled in an 18-footer for birdie thanks to a little help from a playing partner.
'That was a big putt because I got to watch Deane Pappas roll in a 20-footer right in front of me,' acknowledged Mathis.
At the par-four 11th, Mathis hit a gap-wedge to three feet to set up birdie and reach 11-under par for the championship.
Four holes later, Mathis hit a wedge to two feet and kicked in the short birdie putt. Short putts became the theme of the end of the round for Mathis. He birdied the last two holes from three feet and one foot, respectively to get atop the leaderboard.
'I didn't strike the ball as well as I did yesterday, but I hit my wedges really close,' said Mathis, who tied for sixth earlier this month at the South Georgia Classic. 'I worked extremely hard on my wedge game over the winter and it paid off today.'
Matt Bettencourt (67), Peter Tomasulo (69) and Won Joon Lee (65) are tied for fifth place at 9 under par.
Bill Lunde (69), Casey Wittenberg (66), Kim Felton (69), David Branshaw (68) and Roger Tambellini (67) share eighth place at minus-8.
Overnight leader Rick Price struggled to a 2-over 74 at Bright's Creek and fell into a tie for 21st at minus-6.