We examined morphological and diffusion changes of the corpus callosum (CC) in the same group of amputees for the first time. The thickness, area and diffusion tensor imaging (DTI) parameters were used to investigate the CC. Diffusion alteration in the region II of the CC was found in amputees compared with health controls. These changes suggest that fibers connecting bilateral premotor and supplementary motor areas are damaged. The alteration is only reflected in DTI parameters rather than morphological characteristics, indicating that DTI is more sensitive to detect the brain reorgnization following amputation.