The world number 10 was champion of the event in his home state of North Carolina last year and kept his hands on the trophy by beating Berdych 3-6 6-4 7-6 (11/9).
Isner, the 6ft 9in serving menace from Greensboro, fired down 22 aces in the match to follow up his achievement of retaining his grass-court title in Newport, Rhode Island, last month.
As America's leading man in world tennis, the expectation on Isner will be cranked up at the US Open next week.
He found fault in aspects of his performance against Berdych, but to come from a set down to beat the Czech, a former Wimbledon runner-up, was no mean achievement.
Berdych had his match points in the deciding tie-break, but Isner clung on each time.
"It's a great feeling," Isner said on atpworldtour.com.
"Both my titles this year are the exact same titles I won last year. Defending a title is not easy. There's pressure on you coming into the tournament so I think for me to do that, at both Newport and here in Winston-Salem, it should help me a lot going forward in the coming years. I'm absolutely thrilled.
"It was a very good match, an incredible atmosphere. It was a lot of fun to play out there. There were certainly some tense times all throughout the match. At times probably wasn't the prettiest of tennis, but I was able to gut it out and am very, very proud of that."
Isner was a quarter-finalist at Flushing Meadows 12 months ago, the furthest he has ever gone in a grand slam.
"Last year it served me very well," he said. "I won here and made the quarter-finals, lost to Andy Murray in a very tough match.
"I'm heading to New York filled with confidence. I've played very well since my first-round loss [to Alejandro Falla] at Wimbledon. I've only lost three matches, so I'm heading into New York feeling good about myself."