BACKGROUND: Previous studies have tested the association between the rs12722 polymorphism and the risk of anterior cruciate ligament (ACL) injury. Overall, their results are contradictory because most studies used relatively small samples and data from ACL ruptures during sport activities have been mixed with ruptures suffered in non-sporting contexts. OBJECTIVE: To examine the association between the rs12722 polymorphism and the prevalence of ACL rupture in a homogeneous sample of professional male football players. METHODS: A total of 268 professional male football players participated in this study. The rs12722 genotype (CC, CT and TT) was obtained from each player using genomic DNA samples obtained from a buccal swab and measured with PCR RFLP. Players with history of ACL rupture during their professional career were identified by the medical staff of each team. Only ACL injuries obtained during football exposure were considered for this investigation. In this process, we identified 49 ACL ruptures pertaining to 43 players suffered between 2013 and 2024. The situational pattern (i.e., attacking or defending, type of football action, moment of the season, match/training exposure, etc.) was also obtained for each injury. A sub-analysis of non-contact ACL ruptures was conducted, as these injuries are more likely to be influenced by genetic factors. RESULTS: The distribution of genotypes was similar in players with history of ACL rupture (n = 43; CC/CT/TT, 24.4/48.9/26.7%) and with no history of ACL rupture (n = 225; 25.3/49.8/24.9%; = 0.973). Overall, the prevalence of players with history of ACL injury was 16.2% for the whole group of CC players (11 out of 68 players), 16.4% for whole group of CT (22 out of 135 players) and 15.2% for the group of TT players (10 out of 66 players; = 0.973). However, the rs12722 genotype affected the dominance of the injured leg ( = 0.012), the type of action that originated the injury ( = 0.047), and the distribution of non-contact ACL injuries depending on the time of the match ( = 0.020). Specifically, CC players suffered ruptures predominantly in the dominant leg, when landing or reaching (offensive actions) and in the last 15 min of the match (all < 0.050). On the contrary, TT players had ACL ruptures predominantly in their non-dominant leg, when pressing the opponent (defensive actions) and in the first 15 min of the match (all < 0.050). CONCLUSION: There was no association between any of the rs12722 genotypes and the overall prevalence of ACL rupture in professional football players. However, the rs12722 polymorphism appeared to influence specific characteristics of the injury, such as the type of action leading to the rupture and the timing within the match, suggesting a potential genetic contribution to injury susceptibility.