The midfielder was withdrawn in the 18th minute of his country's CONCACAF Gold Cup final clash with Panama on Sunday, which the US went on to win 1-0 courtesy of a Brek Shea goal.
Holden has started just four games in the past two seasons having first been sidelined by an anterior cruciate knee ligament injury before the discovery of cartilage damaged lengthened his recovery period.
And Klinsmann has now hinted that the 27-year-old could be set for more heartache.
"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," the German coach said.
"We'll get an MRI and 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."
Holden joined Bolton Wanders from Houston Dynamo in January 2010, and played a prominent role the following season prior to sustaining his first knee injury against Manchester United.
The US international joined Sheffield Wednesday on a month-long loan toward the back end of the 2012-13 campaign in a bid to prove his fitness.
