The Swedish star limped out of contention at Bethpage Black in Farmingdale after shooting a three-over-par 74 in the first round.
“My right knee, which required surgery in December 2015, has flared up again,” Stenson said in a statement.
“Regrettably, I need to withdraw this week to allow time for an MRI and see what the next step is.”
The world number four's withdrawal raises a question mark about his fitness as Europe prepares to battle the United States at next month's Ryder Cup.
The 40-year-old arrived in New York having won a silver medal in golf's return to the Olympics in Rio de Janeiro.