The 21-year-old, who represented Auburn University, ran 9.84 seconds in one of the quarter-final heats early on Saturday, edging past the previous national mark.
His time beats the long-standing 9.85 seconds set by Olusoji Fasuba back in May 2006.
Ajayi has been closing in on that record for a while.
At the 2025 World Athletics Championships in Tokyo, he ran 9.88 seconds in the heats, becoming the first Nigerian in nearly two decades to reach the 100m final, where he finished sixth.
With this latest run, Ajayi now holds the fastest 100m time recorded globally in 2026.
He moved ahead of Collen Kebinatshipi, who had impressed earlier in the week with a strong showing in the sprint.
Kanyinsola Ajayi is also getting closer to the NCAA record of 9.82 seconds set by Christian Coleman in 2017.
His new time places the Nigerian as the second-fastest collegiate sprinter in U.S. history.
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