The Juventus defender went down after a challenge early in the second half in Poznan and was replaced by his club team-mate Bonucci
The Juventus centre-back was involved in a tangle with striker Kevin Doyle and was immediately replaced by his Bianconero team-mate Leonardo Bonucci.
The Azzurri won the game 2-0 courtesy of goals from Antonio Cassano and Mario Balotelli and have progressed ahead of Croatia and alongside Spain, who have topped the group, to the quarter-final stage, but the injury could be a major loss for Cesare Prandelli's team.
The centre-back had recovered from a thigh injury that he picked up in a win over Atalanta towards the end of the Serie A season, and there were doubts about whether the Old Lady defender would make a full recovery ahead of this summer's tournament.
The substitution was swift and possibly a precaution, but a reoccurrence of the thigh injury could see him miss the rest of the competition.
Goal.com's Italy expert Mark Doyle commented on the Chiellini substitution, saying: "Chiellini being forced off after another tangle with Doyle is very worrying.
"It should not cause too much disruption given his Juventus colleague Bonucci has come on in his place, meaning the Azzurri will still have two Bianconero at the heart of their defence.
"However, Chiellini did not look at all happy and he ever appeared to suggest he's suffered a serious injury, perhaps a break or a muscle tear. That would be a massive blow for the Azzurri given his importance to the team and his versatility."