Gatlin, 34, won a blistering duel with LaShawn Merritt to complete a sprint double following his win in the 100m on the opening weekend at Hayward Field.
Gatlin crossed the line in 19.75 seconds, holding off a late burst from LaShawn Merritt who came home in 19.79sec.
The 2004 Olympic 100m champion later revealed he had been troubled by niggling injuries throughout the meet.
"I've been dealing with ankle and quad injuries through this whole series of races," Gatlin said.
In the women's 200m, reigning Olympic champion Allyson Felix -- who is chasing a 400m-200m double in Rio -- shook off the rust to qualify for Sunday's final.
Felix reached the final after finishing second in her semi behind Tori Bowie with a time of 22.57sec.
"Not as sharp as I'd like to be, but moving on," Felix told reporters.
Merritt, the reigning Olympic champion and world record holder over the distance, had defied medical logic by returning to track and field this season following his kidney transplant last September.
Yet though the 30-year-old clocked his fastest time of the season -- 13.22sec -- it was not enough to earn him a top-three berth and a ticket to Rio.
Merritt later said he planned to protest but admitted that he would have little difficulty putting the disappointment into perspective.
"It's hard not to go back to Rio. But in the circumstances I did the best I could. It's a pity I'm not going to the Games because I know in six weeks time I'll be in much better shape.
Devon Allen won the event in 13.03 seconds from Ronnie Ash
in second and Jeff Porter in third, a fraction ahead of Merritt.
Elsewhere there was a dramatic win in the 5,000m for veteran Kenyan-American Bernard Lagat, who will now be heading to his fifth Olympic games at the age of 41.
"I train with young guys and I don't believe I'm old," Lagat said. "Because if you believe you're old, you're going to run like an old man. These young guys push me every single day.
"What you saw today is what we do in training every day. This was my sixth Olympic trials and out of the six I've made five. This is my greatest achievement.
