Spain's head coach Vicente Del Bosque has omitted Xavi from the Euro 2012 qualifiers against Lithuania and Scotland next month.
The midfielder is understood to be suffering from an ongoing tendinitis problem, although he scored for Barcelona in their weekend win at Athletic Bilbao.
The 30-year-old playmaker joins fellow midfielder Cesc Fabregas and defender Raul Albiol on the injury list for the world champions.
A Barcelona spokesman could not confirm the nature of Xavi's injury.
Fabregas damaged his hamstring during Arsenal's 1-1 draw against Sunderland in mid September and was ruled out of action for two to three weeks.
Meanwhile, Albiol picked up an ankle injury whilst playing for his club, Real Madrid.
Del Bosque's side began their Euro title defence with a 4-0 win in Liechtenstein earlier this month.
They play Lithuania in Del Bosque's home town of Salamanca on October 8th before heading off to face Scotland in Glasgow four days later.
Scotland and Lithuania lead the way in Group I having each collected four points from two games, with Spain a point behind.
"We have two big games, which are both extremely dangerous," said the Spanish coach.
"This is a wake-up call for us. They are two very difficult games.
"The two rivals are ahead of us in the standings, and both are important games in order to achieve qualification for Ukraine and Poland.
"To underestimate them would be dangerous. These are teams that have proven to be difficult and complicated.
"Lithuania are a well-organised team which leave no spaces and has competitive players.
"Scotland, playing at home, will be difficult. We cannot make the slightest mistake. We are prepared for difficult games."
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).