The 28-year-old has only played five times for Wanderers since March 2011, when a tackle by Jonny Evans, which earned the Manchester United defender a red card, saw his knee problems first surface.
His first comeback in September that year lasted just one game and he suffered another setback when he tore his anterior cruciate ligament on international duty in July.
On Monday night Holden took part in a Bolton Under-21s game for the first time since the summer but lasted only 23 minutes before limping off clutching that troublesome right knee.
"It's disappointing news for us all," said Wanderers boss Dougie Freedman.
"We don't know the exact result, (but) we feel that it's the same injury. He's had his scans and he's going to see his specialist on Monday.
"Hopefully he can read the scans and tell us good news, if not he'll need to go into the knee and have a little look."
Holden's contract at the Reebok Stadium is up this summer yet Freedman was adamant that should he finally make a full recovery, his next appearance will not be with another club.
"I went and met him the following night (Tuesday) and had a long chat with him," the Scot added.
"He's very positive and no matter what the outcome of it is, he'll carry on going.
"I want to make it very clear that Stuart Holden will be here in some capacity at this football club - over my dead body is he leaving.
"He's such a fantastic lad and a great ambassador for this football club. You don't need people who are takers; he's never once talked to me about his contract situation, about his (injury) situation.
"He's always talked to me about how can we win games, how can the team get better, how can we get youngsters in the team? It's music to my ears to have people like that around.
"Hopefully he'll be fine and if he's not he'll be back here through the summer getting fit to try and get back in some capacity."
Bolton have always targeted this period in the calendar for Holden's possible return to reserve-team action and Freedman admitted he had been signed off by his surgeons in America to play after three weeks training with the Sky Bet Championship club.
Freedman continued: "He went over to America 10 days ago and they did all the tests and they came up all positive.
"(We were told) three weeks of training and he's ready to play 45 minutes in his first reserve game. Because of Stuart's knowledge of the knee he knew exactly where he was so it wasn't rushed. That wasn't the case. Whether it was bad luck...they'll confirm that for him next week."