Previous studies on gender differences in running biomechanics have predominantly been limited to joint angles and have not investigated a potential influence of footwear condition. This study shall contribute to closing this gap. Lower body biomechanics of 37 recreational runners (19 f, 18 m) were analysed for eight footwear and two running speed conditions. Presenting the effect size Cliff’s Delta enabled the interpretation of gender differences across a variety of variables and conditions. Known gender differences such as a larger range of hip movement in female runners were confirmed. Further previously undiscovered gender differences in running biomechanics were identified. In women, the knee extensors are less involved in joint work. Instead, compared to men, the supinators contribute more to deceleration and the hip abductors to acceleration. In addition to differences in extent, women also show a temporal delay within certain variables. For the foot, ankle and shank, as well as for the distribution of joint work, gender differences were found to be dependent on footwear condition, while sagittal pelvis and non-sagittal hip and thigh kinematics are rather consistent. On average, smaller gender differences were found for an individual compared to a uniform running speed. Future studies on gender differences should consider the influence of footwear and running speed and should provide an accurate description of the footwear condition used. The findings of this study could be used for the development of gender-specific running shoes and sports and medical products and provide a foundation for the application of smart wearable devices in gender-specific training and rehabilitation.