The corpus callosum (CC) has been implicated in the reorganization of the brain following amputation. However, it is unclear which regions of the CC are involved in this process. In this study, we explored the morphometric and microstructural changes in CC subregions in patients with unilateral lower limb amputation. Thirty-eight patients and 38 age- and gender-matched normal controls were included. The CC was divided into five regions, and the area, thickness and diffusion parameters of each region were investigated. While morphometric analysis showed no significant differences between the two groups, amputees showed significant higher values in axial diffusivity, radial diffusivity and mean diffusivity in region II of the CC, which connects the bilateral premotor and supplementary motor areas. In contrast, the mean fractional anisotropy value of the fibers generated by these cortical areas, as measured by tractography, was significantly smaller in amputees. These results demonstrate that the interhemispheric pathways contributing to motor coordination and imagery are reorganized in lower limb amputees.