Liverpool's Joe Allen will miss Wales' World Cup qualifiers against Macedonia and Serbia.
Midfielder Allen was injured playing for his club against Notts County in the 4-2 win at Anfield on Tuesday night in the Capital One Cup.
Allen joins Swansea City's Neil Taylor on the sidelines.
They will be replaced in Chris Coleman's squad by David Vaughan of Sunderland and Ashley Richards of Swansea City.
There were fears over Aaron Ramsey's fitness after the midfielder suffered a groin injury late on in Arsenal's Champions League win over Fenerbahce.
Ramsey finished the match on the bench but is expected to be available for Sunday's North London derby against Tottenham.
Fellow midfielder Allen limped off with a hamstring injury during Liverpool's victory over Notts County.
Defender Taylor is not included after being ruled out for four to six weeks after suffering severe bruising on his thigh during Swansea's Europa League win over Petrolul Ploiesti last week.
There is no place in the squad for West Ham defender James Collins and Sunderland midfielder David Vaughan but Brighton's Andrew Crofts and Birmingham City full-back Neal Eardley are recalled.
Manager Chris Coleman has included Gareth Bale in his squad as speculation over a proposed world-record transfer from Tottenham to Real Madrid continues to intensify.
Wayne Hennessey (Wolverhampton Wanderers - on loan at Yeovil), Boaz Myhill (West Bromwich Albion), Owain Fon Williams (Tranmere Rovers), Ben Davies (Swansea City), Neal Eardley (Birmingham City), Danny Gabbidon (Crystal Palace), Chris Gunter (Reading), Adam Matthews (Celtic), Sam Ricketts (Wolverhampton Wanderers), Ashley Williams (Swansea City), Andy King (Leicester City), Joe Ledley (Celtic), Aaron Ramsey (Arsenal), Jonathan Williams (Crystal Palace), David Vaughan (Sunderland), Jack Collison (West Ham United), Andrew Crofts (Brighton & Hove Albion), Sam Vokes (Burnley), Craig Bellamy (Cardiff City), Simon Church (Charlton Athletic), Hal Robson-Kanu (Reading), Gareth Bale (Tottenham), Ashley Richards (Swansea City)