Gatlin upstaged the Jamaican with the top time in the first round, even though the American got a little push from nature with a strong back wind of 2.1 meters per second during his 9.83-second race.
Bolt could count on an adoring crowd but faced a slight headwind as he cruised to a time of 9.96, the fifth fastest overall.
"It was OK. It was not perfect," Bolt said. "I still have some adjustments. Just have to concentrate on my technique now."