Trayvon Bromell, 19, equalled the third fastest 100 in the world this year by running 9.84 seconds to top qualifying.
Gay's training partner, Remontay McClain, was even faster, the 22-year-old clocking 9.82 seconds though he was aided by a 4.9 metres per second wind, over the allowable limit.
The 32-year-old Gay, who missed a year of competition for doping in 2013, ran in the same heat as McClain and took second place in 9.85.
The 2007 world champion said it had been a different feeling competing with the new American crop.
"It was weird, maybe weird is not the right word, against a group of guys I am running against now," said Gay.
"It is a different era for me. All the new faces it was just different.
"I just wanted to get through as easy as possible," he added. "I felt relaxed, got the nerves out of way."
The women's heats were also impressive but windy, with Olympic champion Carmelita Jeter the fastest at 10.87 seconds with an assisting wind of 4.5 metres per second.
The semi-finals and finals of the men's and women's 100 are scheduled for Friday.
The top three finishers in each event at the trials qualify for the world championships in Beijing in August.
(Reporting by Gene Cherry in Eugene, Oregon; Editing by Peter Rutherford)
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