US Soccer announced that Holden, who plays for England's Bolton Wanderers, was found to have a torn anterior cruciate ligament and that he will consult Bolton and US Soccer doctors to devise a treatment plan.
"We are absolutely devastated for Stuart," said US coach Jurgen Klinsmann. "He is such a great part of our team on the field and the locker room. He worked tremendously hard to recover from previous injuries and had really come back into form.
"He was fully prepared to head back to Bolton and challenge for a starting spot. Now he will have our full support as he goes down this road again, and we will be with him every step of the way."
Holden suffered the injury in the 18th minute of the 1-0 US victory over Panama at Chicago's Soldier Field.
He had recently returned to the national squad, making his first appearance in more than two years as a substitute in a 29 May loss to Belgium.
The Americans, on a team-record 11-match win streak, have not lost since.