Barcelona playmaker, who is said to suffer from tendinitis, joining fellow midfielder Cesc Fabregas and defender Raul Albiol on the injury list.
Fabregas damaging a hamstring during Arsenal's 1-1 draw with Sunderland on September 18.
He is expected to be out for two to three weeks.
Albiol misses out despite returning to training with Real Madrid this month after a ligament injury to take over the summer.
Meanwhile, Barcelona's spokesman could not immediately confirm the nature of the injury Xavi despite media reports blamed the tendinitis in his homeland.
World champions Spain began to defend their European title with a 4-0 victory in Liechtenstein earlier this month.
They play Lithuania in Salamanca on October 8, and Scotland in Glasgow four days later in the final round of Group I game.
Scotland led the group from Lithuania thanks to goals scored, with Spain turning point but with a game in hand.
Spain squad
Goalkeepers: Iker Casillas (Real Madrid), Victor Valdes (Barcelona), Pepe Reina (Liverpool)
Defenders: Alvaro Arbeloa (Real Madrid), Sergio Ramos (Real Madrid), Carles Puyol (Barcelona), Gerard Pique (Barcelona), Carlos Marchena (Villarreal), Joan Capdevila (Villarreal), Nacho Monreal (Osasuna)
Midfielders: Xabi Alonso (Real Madrid), Sergio Busquets (Barcelona), Santi Cazorla (Villarreal), Andres Iniesta (Barcelona)
Forwards: Pedro (Barcelona), Jesus Navas (Sevilla), David Villa (Barcelona), David Silva (Manchester City), Fernando Llorente (Athletic Bilbao), Fernando Torres (Liverpool).
source : BBC
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