Fully fit for the first time in years, Gay took control mid-race with a dominant run to equal the 10th fastest 100m of all time. Gatlin finished second in 9.89 seconds in the race at Des Moines, Iowa.
"It's been five years since I have been the US champion and it feels good to be back on the podium," said Gay, beset by injuries since winning both sprint titles at the 2007 world championships.
"I'm not as sharp as I could be but I'm healthy so that's all that counts," said Gay, who ran another 9.75, this one wind-assisted, in the semi-finals.
Gatlin, who surprised Jamaican world record holder Usain Bolt in Rome earlier in the month, said he was affected by a strained right hamstring.
"It felt good running 9.8 with a bandage on," said the 2004 Olympic gold medallist who narrowly edged Gay for the bronze medal at last summer's London Games.
Charles Silmon grabbed the third spot on the US team for the Aug. 10-18 world championships by edging Mike Rodgers by two thousandths of a second. Silmon ran 9.972 and Rodgers 9.974.
Collegiate champion English Gardner came on strong to win the women's 100 in another year-best time, 10.85 seconds, and Olympian Sharon Day delivered the top heptathlon score of the season, 6,550 points.
Gardner, now coached by John Smith, edged Octavious Freeman (10.87) with Alexandria Anderson grabbing third (10.91).
Haitian-born Barbara Pierre struggled to finish fifth after setting the world lead of 10.85 in the semi-finals that Gardner equalled.
The 100m hurdlers also flexed their speed, with collegiate record holder Brianna Rollins blitzing to an eye-catching but wind-assisted 12.33 seconds in qualifying.
Queen Harrison added a wind-assisted 12.44 seconds, hometown Olympian Lolo Jones clocked a wind-allowable 12.50 seconds.
The 2008 Beijing gold medallist Dawn Harper won her preliminary in a wind-assisted 12.60.
World record holder Ashton Eaton, taking a "play it safe" approach, trailed Gunnar Nixon after the first day of the decathlon with 4,405 points. Nixon had 4,449.
Beijing Olympic gold medallist Lashawn Merritt led 400m qualifying in 44.36 seconds but London winner Sanya Richards-Ross, hampered by toe surgery, barely slipped into Saturday's women's final as the next-to-last qualifier.
She ran 51.53 with Francena McCorory the leader at 50.53.
The trials continue through to Sunday.
1. Tyson Gay 9.75
2. Justin Gatlin 9.89
3. Charles Silmon 9.98
Men's Pole Vault
1. Brad Walker 5.65
2. Jeremy Scott 5.65
3. Jack Whitt 5.60
Men's Discus Throw
1. Lance Brooks 62.29
2. Russ Winger 62.03
3. James Plummer 61.96
Men's Hammer Throw
1. A G Kruger 75.52
2. Chris Cralle 74.55
3. Andrew Loftin 73.63
1. English Gardner 10.85
2. Octavious Freeman 10.87
3. Alexandria Anderson 10.91