The Barcelona defender missed the world and European champions' training session on Saturday and may not feature in Monday's Group B clash in Curitiba.
A statement on the official website for the Spanish Football Federation (RFEF) said: "Gerard Pique had to withdraw due to pain in the adductor muscle region."
Pique was dropped for Spain's 2-0 defeat against Chile on Wednesday on the back of their 5-1 thrashing at the hands of the Netherlands in their first game of the tournament.
Spain and Australia will only have pride to play for in Curitiba, having both failed to pick up a solitary point from their first two games.
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