The 27-year-old suffered his latest setback representing the United States in Sunday's Gold Cup final against Panama.
Holden sustained a similar injury against Manchester United back in 2011, and after a six-month absence required further surgery following a League Cup clash with Aston Villa.
United States coach Jurgen Klinsmann told ussoccer.com: "We are very concerned about Stuart's situation. It looks like a very serious injury, but I'm not a doctor and I can't confirm it.
"He'll have all the tests done. Stu is a player that when something happens, he knows when something happens. Hopefully it's not as bad, but it's not looking good right now."