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Yes a number 16 seed has beat a number one seed in the NCAA Basketball tournament, but it was in the womens' tournament. In 1988, 16th seed Harvard beat 1st seed Stanford in the womens' tourney. (Answer by Mymomisnfat)

It also happened in 2018 when the University of Maryland - Baltimore County (16th seed in the South) beat the University of Virginia (1st seed in the South) by a score of 74 - 54.

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