Gentiletti - a new recruit after winning the Copa Libertadores with Argentina's San Lorenzo last month - injured the anterior cruciate ligament in his left knee during Lazio's 1-0 Serie A loss to Genoa on Sunday.
Lazio's worst fears were confirmed post-game, with the club confirming the 29-year-old will "undergo surgery in the coming days".
After missing Lazio's opening league fixture against Milan, Gentiletti started back-to-back matches against Cesena and Genoa.
Argentinian midfielder Lucas Biglia (foot) and Serbian utility Dusan Basta (hand) also sustained injuries on Sunday.
Lazio announced both players will undergo further tests.
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