The two 22-year-olds have played mostly for the under-21 side since taking part in Spain's successful World Cup campaign in South Africa last year.
Spain play the Czechs on March 25 and travel to Lithuania four days later.
Valencia's Mata comes in as cover for injured Barcelona forward Pedro while Athletic Bilbao's Martinez replaces Arsenal's Cesc Fabregas, who has a hamstring problem.
Athletic Bilbao defender Andoni Iraola was named to fill the spot vacated by injured Barcelona captain Carles Puyol.
Spain top their group on nine points after three wins from three matches. The Czechs have six points and Scotland and Lithuania are level on four. Liechtenstein are bottom of the five-nation standings with zero points.
Coach Vicente Del Bosque said he was concerned that many members of the squad, particularly those from Barcelona and Real Madrid, were fighting on three fronts in domestic and European competition.
Barca top La Liga ahead of Real, while the great rivals play each other in next month's King's Cup final and could meet in the Champions League semi-finals.
"We are worried as the majority of our players are competing for everything around the end of the season," Del Bosque told a news conference.
"In between there is a slot for the national team with two very strong opponents, especially Czech Republic, who are our biggest threat given the points they have," he added.
"Our main concern is to be as mentally focused as possible because anything else would make our task tougher."
Goalkeepers: Iker Casillas (Real Madrid), Victor Valdes (Barcelona), Pepe Reina (Liverpool).
Defenders: Alvaro Arbeloa (Real Madrid), Sergio Ramos (Real Madrid), Raul Albiol (Real Madrid), Gerard Pique (Barcelona), Carlos Marchena (Villarreal), Joan Capdevila (Villarreal), Andoni Iraola (Athletic Bilbao).
Midfielders: Xavi (Barcelona), Xabi Alonso (Real Madrid), Andres Iniesta (Barcelona), Javi Martinez (Athletic Bilbao), Sergio Busquets (Barcelona), Santi Cazorla (Villarreal).
Forwards: David Silva (Manchester City), David Villa (Barcelona), Fernando Torres (Chelsea), Fernando Llorente (Athletic Bilbao), Jesus Navas (Sevilla), Juan Mata (Valencia).
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