Real Madrid forward Gareth Bale will miss Wales' friendly against Holland on November 13.
Bale has not played since October 17 after suffering a recurrence of a calf injury following the October internationals.
The 26-year-old featured in the first half of Real's 3-0 victory over Levante four days after Wales' Euro 2016 qualification party in Cardiff.
Bale was criticised by some Madrid journalists and fans for playing in Wales' 2-0 victory over Andorra after previously being sidelined for nearly three weeks with a calf strain.
Wales boss Chris Coleman was also criticised by Arsenal manager Arsene Wenger for playing Aaron Ramsey against Andorra with Wales having already qualified for next summer's European Championship.
Ramsey subsequently picked up a hamstring injury on Arsenal duty and also misses Wales' first Euro 2016 warm-up game at the Cardiff City Stadium.
Reading forward Hal Robson-Kanu and Fulham defender Jazz Richards are other absentees through injury.
Newcastle defender Paul Dummett and Walsall striker Tom Bradshaw have been called into the squad.
Wales squad: Hennessey (Crystal Palace), Fon Williams (Inverness), Ward (Liverpool); A Williams (Swansea, capt), Chester (West Brom), Gunter (Reading), Taylor (Swansea), Davies (Tottenham), Collins (West Ham), Henley (Blackburn), Dummett (Newcastle), Vaughan (Nottingham Forest), Huws (Wigan), Allen (Liverpool), Ledley (Crystal Palace), King (Leicester), Edwards (Wolves), J Williams (Crystal Palace), Cotterill (Birmingham), Lawrence (Leicester), Church (MK Dons), Vokes (Burnley), Bradshaw (Walsall).