The purpose was to examine (1) the effect of measurement position and sex on femoral cartilage outcomes, and (2) the association between gait biomechanics and cartilage outcomes. Fifty individuals participated (25 males and 25 females; age = 20.62 [1.80] y). Ultrasound measured femoral cartilage thickness and echo-intensity at 90°, 115°, and 140° of knee flexion. Gait outcomes included the external knee adduction and knee flexion moments. Cartilage outcomes were compared using 2 (sex) × 3 (position) repeated-measures analysis of variance. Gait and cartilage associations were assessed using stepwise regression. Medial cartilage was thicker when measured at 90° compared with 115° (P = .02) and 140° (P < .01), and 115° compared with 140°, (P < .01) in males but not in females. Cartilage was thicker at 90° compared with 140° across both sexes within all regions (P < .01). Males had thicker cartilage than females in all positions (P < .01). Echo-intensity was lower at 90° than 115° (P < .01) and 140° (P = .01) in the central and lower at 90° than at 115° (P < .01) and 140° (P = .03) in lateral regions. No association was found between gait and cartilage outcomes. Ultrasound imaging position effects cartilage features more in males compared with females. Imaging position and sex influence cartilage outcomes and should be considered in study designs and clinical evaluation.