Serena Williams withdrew from the Western & Southern Open ahead of her opening match due to the back injury she suffered in Toronto.
Williams, 37, was due to face Zarina Diyas in Cincinnati on Tuesday, but pulled out as the American great continues to struggle with her back.
The 23-time grand slam champion retired during the Rogers Cup final against Bianca Andreescu on Sunday due to back spasms.
"I came to Mason on Sunday and have tried everything to be ready to play tonight, and was still hopeful after my practice this morning," two-time champion Williams said, via the tournament's Twitter account.
"But unfortunately my back is still not right."
Williams' withdrawal is a concern with the US Open less than two weeks away and the veteran still aiming to equal Margaret Court's record of 24 grand slam titles.
Jessica Pegula, the Citi Open champion, has taken Williams' place and will face Diyas, who knocked her out in qualifying.