Chris Mepham has withdrawn from the Wales squad for the Nations League clash against Denmark on Friday.
Sheffield United's Kieron Freeman has been called up to replace him for the Denmark game at Cardiff and next Tuesday's friendly with Albania in Elbasan.
Brentford defender Mepham pulled out of last month's high-profile friendly against Spain and the Nations League tie with the Republic of Ireland after picking up a knock in training.
Mepham has played four times for the Bees since the last international break, but it is understood he has suffered a recurrence of the unspecified injury that ruled him out last month.
Press Association Sport understands Mepham was ruled out after having the injury scanned and has returned to Brentford for treatment.
Mepham, 20, has impressed since making his international debut at the China Cup in March.
The London-born centre-back started both Nations League games against the Republic and Denmark in September.
Blades defender Freeman was drafted into the Wales squad for the Dublin clash last month.
The Nottingham-born 26-year-old has played at under-17, under-19 and under-21 level for Wales but has yet to make an appearance for the senior team.
Meanwhile, the Football Association of Wales have declared that Friday's potential group decider at the Cardiff City Stadium is a 33,000 sell-out.
