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The 14-time Olympic gold medalist swam the two-lap final in 50.22 seconds at the Indiana University Natatorium on Thursday night. He lowered Ian Crocker's mark of 50.40 set at the 2005 world championships in Montreal.
Phelps had once owned the 100 fly mark for a day at the 2003 world meet in Barcelona. But Crocker took it from him a day later, then lowered it twice more.
Phelps' victory gave him a spot in the event at the world championships later this month in Rome. He already qualified in the 200 fly and 200 freestyle as he continues his transformation from swimming middle distances to sprints.
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