AbstractThe COL5A1 and COL12A1 variants are independently associated with modulating the risk of anterior cruciate ligament (ACL) rupture in females. The objective of this study was to further investigate if COL3A1 and COL6A1 variants independently, as well as, collagen gene–gene interactions, modulate ACL rupture risk. Three hundred and thirty‐three South African (SA, n = 242) and Polish (PL, n = 91) participants with diagnosed ACL ruptures and 378 controls (235 SA and 143 PL) were recruited. Participants were genotyped for COL3A1 rs1800255 G/A, COL5A1 rs12722 (T/C), COL6A1 rs35796750 (T/C) and COL12A1 rs970547 (A/G). No significant associations were identified between COL6A1 rs35796750 and COL3A1 rs1800255 genotypes and risk of ACL rupture in the SA cohort. The COL3A1 AA genotype was, however, significantly (p = 0.036) over‐represented in the PL ACL group (9.9%, n = 9) when compared to the PL control (CON) group (2.8%, n = 4). Although there were genotype distribution differences between the SA and PL cohorts, the T+A‐inferred pseudo‐haplotype constructed from COL5A1 and COL12A1 was significantly over‐represented in the female ACL group when compared to the female CON group within the SA (T+A ACL 50.5%, T+A CON 38.1%, p = 0.022), PL (T+A ACL 56.3%, T+A CON 36.3%, p = 0.029) and combined (T+A ACL 51.8%, T+A CON 37.5%, p = 0.004) cohorts. In conclusion, the novel main finding of this study was a significant interaction between the COL5A1 rs12722 T/C and COL12A1 rs970547 A/G variants and risk of ACL injury. These results highlight the importance of investigating gene–gene interactions in the aetiology of ACL ruptures in multiple independent cohorts.