Ambrose, racing for the Richard Petty team, won the top spot in Sunday's Cheez-It 355 by covering the 2.45-mile course in 128.241 miles per hour.
He cracked the previous track record of 127.020 mph set in 2010 by Juan Pablo Montoya.
Road courses have been kind to Ambrose. He has won the past two Watkins Glen events and was on the pole at last year's road race in Sonoma, Calif.
Pregnant Mila Kunis wins 'Best Villain' at MTV Movie Awards
Biologists detail four new deep-sea 'killer sponges'