The defeat marked the first time that Williams has fallen in the first round of the U.S. Open singles draw.
Venus Williams made her 22nd women's singles U.S. Open appearance this year, the most all-time among women. But on Tuesday night, Williams lost in straight sets, 6-3, 7-5, to Karolina Muchova on Tuesday.
The defeat marked the first time that Williams, 40, has fallen in the first round of the U.S. Open singles draw.
Williams, who won her second Grand Slam title at the U.S. Open in 2000, has not advanced past the third round of the U.S. Open singles draw since 2017, when she made the semifinals.
In tennis' first major championship of the year, the Australian Open, Williams also lost in the first round.
Venus' defeat comes as her sister, Serena Williams, also made history in Flushing Meadows, Queens on Tuesday. The younger Williams sister broke a U.S open record for most career wins in the tournament's history, male or female, after defeating Kristie Ahn in straight sets.
Serena is looking to win her 24th major title this year and her first major championship since winning in Australia in 2017. She is set to face off against Margarita Gasparyan on Thursday.