The Englishman produced a flawless, bogey-free display, racking up eight birdies — highlighted by a stunning 36-foot putt on the 15th — at Hilton Head.
Fitzpatrick, the 2022 US Open champion, had started the day in a six-way tie for third after opening with a 65, but his latest round lifted him to 14 under par and into outright first place.
Norway's Viktor Hovland remains in close pursuit, just one shot back, after backing up his strong start with another 65 to hold onto second position.
Overnight leader Ludvig Aberg slipped down the standings to a share of fourth after stumbling with three bogeys on the back nine.
Scotland's Robert MacIntyre sits six shots adrift following a round of 68, while world number one Scottie Scheffler posted a 67 to leave himself seven off the lead heading into the weekend.
Matt Fitzpatrick and his younger brother Alex held their nerve down the stretch to secure a landmark win at the Zurich Classic of New Orleans, a triumph that also hands Alex a two-year PGA Tour exemption.
Star pairing Brooks Koepka and Shane Lowry fell short of the leaders at the Zurich Classic of New Orleans, missing the cut by a single shot as they exited before the weekend.
Matt Fitzpatrick held his nerve to edge Scottie Scheffler on the first playoff hole in a thrilling finish at the RBC Heritage on Sunday.
Scottie Scheffler surged into contention at the PGA Tour's RBC Heritage on Saturday, but Matt Fitzpatrick still leads the tournament by three shots.
