Knowledge of the morphology and kinematics of the male and female knees is important for understanding gender-related dimorphism in knee pathology and improvement of related surgical treatments. Twelve male and 12 female subjects with healthy knees were recruited, and each subject performed a single leg lunge while images of the knee were recorded by two fluoroscopes. Tibiofemoral joint motion was then reproduced using bony models matched to the fluoroscopic images. Femoral trochlear groove orientation was also measured in each knee. While many of the measured parameters were found to be similar between the genders, a few interesting differences were also noted. Females showed greater external tibial rotation at 0 degrees flexion (-5.4 degrees vs. -1.3 degrees, p = 0.03), smaller internal rotation at 30 degrees flexion (1.7 degrees vs. 6.4 degrees, p = 0.04) and greater range of tibial rotation (18.2 degrees vs. 12.4 degrees, p = 0.01) compared to males. Female knees also had a more medially oriented proximal trochlear groove (10.0 degrees vs. 4.5 degrees, p = 0.04). These gender differences in rotational kinematics and trochlear groove orientation may warrant further studies to determine implications for surgical treatments such as total knee arthroplasty, and gender-related dimorphism in certain knee injuries and pathologies, like anterior cruciate ligament injury and patellofemoral problems.