[Purpose] We investigated the difference in onset time between the vastus medialis and lateralis according to knee alignment during stair ascent and descent to examine the effects of knee alignment on the quadriceps during stair stepping. [Subjects] Fifty-two adults (20 with genu varum, 12 with genu valgum, and 20 controls) were enrolled. Subjects with > 4 cm between the medial epicondyles of the knees were placed in the genu varum group, whereas subjects with > 4 cm between the medial malleolus of the ankle were placed in the genu valgum group. [Methods] Surface electromyography was used to measure the onset times of the vastus medialis and vastus lateralis during stair ascent and descent. [Results] The vastus lateralis showed more delayed firing than the vastus medialis in the genu varum group, whereas vastus medialis firing was more delayed than vastus lateralis firing in the genu valgum group. Significant differences in onset time were detected between stair ascent and descent in the genu varum and valgum groups. [Conclusion] Genu varum and valgum affect quadriceps firing during stair stepping. Therefore, selective rehabilitation training of the quadriceps femoris should be considered to prevent pain or knee malalignment deformities.