The Manchester City striker had been set to return to the international fold after a three-game absence but the 30-year-old has been advised not to travel by medical staff
Bellamy is the seventh player to pull out of Sunday's game in Osijek following James Collins, Danny Gabbidon, Simon Davies, Andy King, David Cotterill and Ched Evans.
John Toshack has promoted four of the Under-21 squad to his senior party in Mark Bradley, Neil Taylor, Christian Ribeiro and Hal Robson-Kanu.
Wales already had considerable injury problems with Aaron Ramsey, Jack Collison, Gareth Bale and Joe Allen all out while players from Championship play-off finalists Cardiff and Blackpool have not been considered.
The absences leave Wales with a squad of just 18 to travel to Croatia tomorrow.
Bellamy's initial selection had been a surprise after Toshack previously suggested he might not select his skipper for friendlies.
Bellamy was overlooked for the last fixture against Sweden in March despite being fully fit, prompting speculation about the player's international future.
Toshack refused to confirm whether his talismanic striker would return for the next competitive fixtures in the autumn but his potential return for Croatia had suggested any problems had been resolved.