Background The classification of knee osteoarthritis is an essential clinical issue, particularly in terms of diagnosing early knee osteoarthritis. However, the evaluation of three-dimensional limb alignment on two-dimensional radiographs is limited. This study evaluated the three-dimensional changes induced by weight-bearing in the alignments of lower limbs at various stages of knee osteoarthritis. Methods Forty five knees of 25 patients (69.9 +/- 8.9 years) with knee OA were examined in the study. CT images of the entire leg were obtained in the supine and standing positions using conventional CT and 320-row detector upright CT, respectively. Next, the differences in the three-dimensional alignment of the entire leg in the supine and standing positions were obtained using 3D-3D surface registration technique, and those were compared for each Kellgren-Lawrence grade. Results Greater flexion, adduction, and tibial internal rotation were observed in the standing position, as opposed to the supine position. Kellgren-Lawrence grades 1 and 4 showed significant differences in flexion, adduction, and tibial internal rotation between two postures. Grades 2 and 4 showed significant differences in adduction, while grades 1 and 2, and 1 and 3 showed significant differences in tibial internal rotation between standing and supine positions. Conclusions Weight-bearing makes greater the three-dimensional deformities in knees with osteoarthritis. Particularly, greater tibial internal rotation was observed in patients with grades 2 and 3 compared to those with grade 1. The greater tibial internal rotation due to weight-bearing is a key pathologic feature to detect early osteoarthritic change in knees undergoing osteoarthritis.