The Juventus star limped out of a training session on Tuesday with a swollen calf.
Italy's team doctor Enrico Castellacci has subsequently confirmed he will not be risked for the friendly match with Netherlands in Bari, but is hopeful the 30-year-old can feature in Italy's opening Euro 2016 qualifying clash in Norway.
"Chiellini has some pain in his left calf," he said. "This morning we performed an ultrasound that showed some swelling.
"There's no damage to the muscle fibres, but just fatigue. He will miss the match on Thursday. We will try and help him recover for Tuesday."
Italy earlier confirmed that Inter forward Pablo Daniel Osvaldo (hamstring) and Parma defender Gabriel Paletta (back) will both miss out through injury.
Elsewhere, Stephan El Shaarawy is delighted to be back playing after missing almost all of the previous campaign through injury.
The Milan forward has thanked club boss Filippo Inzaghi and new Italy coach Antonio Conte for retaining confidence in his ability.
"I'm glad to be back playing," he said. "I'm feeling really good, physically.
"Inzaghi and Conte have given me so much. I hope to repay their confidence. They have both made me understand that I have ability to do well.
"Both are motivators and can express what they want to the group."
Conte is set to take charge of Italy for the first time since being named as Cesare Prandelli's successor last month.
