The four-time 200m world champion American pushed Cameroon’s Emmanuel Eseme (9.94) into second with Letsile Tebogo (9.95) of Botswana in third.
Italian former Olympic champion Marcell Jacobs came fifth in 9.99sec.
Lyles, 28, had won his first race of the season in Tokyo two weeks ago in 9.95sec.
Sri Lanka’s Rumesh Tharanga Pathirage produced a stunning 92.62m in the javelin to propel himself into eighth on the all-time list in the event.
The 23-year-old had already thrown the year’s best distance of 89.37m in March but he still has a few more metres to find to trouble Jan Zelezny’s world record of 98.48m back in May 1996.
“I tried my best today to throw the national record, I managed to improve it by 30cm, even though I had only two valid attempts today,” said Pathirage.
“The weather feels good in Rome to throw further than at the last competition (in Rabat). Winning today feels like a Sri Lankan festival.”
Anderson Peters of Grenada came second with 83.91m, just 2cm ahead of American Curtis Thompson in third.
Olympic silver-medallist Julien Alfred upset world champion Melissa Jefferson-Wooden in the women’s 200m, clocking 21.93sec.
Alfred of Saint Lucia has posted the fastest 200m time this season with a 21.86sec.
American Jefferson-Wooden, who also won the 100m and 4x100m in Tokyo last year, was racing a 200m for the first time in 2026.
She finished second in 22.17 with compatriot Anavia Battle third in 22.39. (AFP)