The American will be out of action for between six to nine months after undergoing an operation on the knee ligaments he injured in an U21 match against Everton last week.
That match was his first since he recovered from tearing his anterior cruciate ligament in his right knee during the USA's CONCACAF Gold Cup final win over Panama last July.
Bolton boss Dougie Freedman said: "Unfortunately, Stuart's knee has gone. His surgeon has had a look and has deemed this to be the best course of action.
"We're all obviously very disappointed for Stuart, but he's a warrior and a fighter and someone who knows exactly what he wants to achieve. Everyone at the football club is right behind him."
Holden's terrible run of injuries began in 2010 when he broke his leg playing for the USA against the Netherlands.
Then in March 2011 he suffered a serious knee injury in a tackle with Manchester United's Jonny Evans which kept him out for over a year.