Gay overcame a slow start to pull away from Powell, who clocked 9.90. American Darvis Patton finished third in 10.00, with world champion and world record-holder Usain Bolt skipping the race.
"It was a tough race," Gay said. "I think Asafa kind of relaxed ... I didn't quit the race, even though I was far behind him."
The American has beaten Powell four times this season, with the Jamaican winning once.
enthusiastic applause
Bolt, who smashed the 100 and 200 records at last month's World Championships in Berlin, will race in the 200 metres today.
Yesterday, the Jamaican star ran on to the track to enthusiastic applause from a crowd of 20,000 at Kaftanzoglio stadium.
Jamaica's world champion Brigitte Foster-Hylton won the women's 100 metres hurdles. She clocked 12.58 seconds to beat American Dawn Harper who finished second in 12.61 seconds, and fellow Jamaican Delloreen Ennis-London who was third in a similar time.
Allyson Felix won the women's 200, beating fellow American Sanya Richards in a photo finish after both runners clocked 22.29 seconds. Kerron Stewart of Jamaica was third in 22.42.
In the women's pole vault, world record-holder Yelena Isinbayeva cleared a modest 4.80 metres for an easy win.
The Russian star - who has broken the world record 27 times in her career - then had a failed attempt at 5.07 metres. Fabiana Murer of Brazil and Poland's Monika Pyrek shared second place with 4.60.
Isinbayeva said her win felt like "revenge" for her surprise loss last month in Berlin when she failed to clear a height.
unbeaten streak
She rebounded days later to set a new world record of 5.06 in Zürich. She said yesterday her aim is set much higher in the future.
"I still think that a 5.20-metre jump is possible," she said. "This season was very up and down. ... I tried my best today."
In the 400, Olympic and world champion LaShawn Merritt of the United States won in 44.93 seconds. Chris Brown of The Bahamas clocked 45.49 in second and American David Neville came third in 45.60.
The 23-year-old Merritt extended his unbeaten streak to 12 races, but was unhappy with his time after clocking 44.06 at the Worlds.
"All that matters in these races is victory. I think I can be called the best 400-metre runner (this season) - I've earned it."
Brittney Reese of the United States won the long jump with 7.08 metres, pushing Russian duo Elena Sokolova and Tatyana Lebedeva to second and third place with jumps of 6.81 and 6.79, respectively.
Javelin world champion Steffi Nerius of Germany announced the end of her international career after throwing 62.59 for third place.
"The journey ends here," the 37-year-old Nerius said. "All these years I won many medals - Olympic, world, European. I achieved everything I could dream of."
Nerius wore her trademark headband with the words 'Goodbye' written in English, Greek and German.
Maria Abakumova of Russia won the event, throwing 64.63, while world record-holder Barbora Spotakova of the Czech Republic was third with 63.45.