This study investigated the effect of locomotive organ dysfunction on physical fitness in third- to sixth-grade school students. The effect was evaluated through musculoskeletal examination, and physical fitness was measured through a physical fitness test. In total, 884 third-to sixth-grade students (466 boys and 418 girls; mean age: 11.1 ± 0.86 years; height: 142.0 ± 8.82 cm, bodyweight: 34.8 ± 8.00 kg) underwent musculoskeletal examination of their locomotive organs and completed a physical fitness test. The difference between the physical fitness test scores and average total scores of the locomotive and nonlocomotive groups showed a significant primary effect of the locomotive factors in the upper body lifts, repeated side jumps, 20 m shuttle run, and standing long jumps. (F = 5.71-9.88, p < 0.05). These results, indicate that the physical fitness in children who tested positive for locomotive organ dysfunction was significantly lower than that in children without locomotive disorders.