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Objective: Ankle joint line orientation (AJLO) is influenced by the subtalar foot and frontal leg alignment. However, the influence of axial leg alignment on AJLO remains unclear. The study aimed to analyze the influence of frontal, axial leg alignment on AJLO in healthy subjects. Methods: Thirty healthy subjects (60 legs) without prior surgery underwent standing biplanar long leg radiograph (LLR) between 2016 and 2020. AJLO was measured in standing long‐leg radiographs relative to the ground. Meary's angle and calcaneal pitch were measured. Hip‐knee‐ankle angle (HKA), femoral antetorsion, and tibial torsion were assessed with SterEOS software (EOS Imaging, Paris, France). LLR was acquired with the feet directing straight anteriorly, which corresponds to a neutral foot progression angle (FPA). The influence of subtalar, frontal, and axial alignment on AJLO was analyzed in a multiple regression model. Results: An increase in knee valgus increased relative valgus AJLO by 0.5° (95% CI: 0.2° to 0.7°) per 1° (P < 0.001). A decrease in femoral antetorsion increased relative valgus AJLO by 0.2° (95% CI: 0.1° to 0.2°) per 1° (P < 0.001), whereas Meary's angle and calcaneal pitch did not influence AJLO. Conclusion: A link between frontal, axial leg alignment, and AJLO could be demonstrated, indicating that a valgus leg alignment and relative femoral retrotorsion are associated with an increase of valgus AJLO in healthy subjects when placing their feet in a neutral position. Alteration of the frontal, or rotational profile after realignment surgery or by implant positioning might influence the AJLO, when the FPA is kept constant. [ABSTRACT FROM AUTHOR]