Diffusion tensor imaging (DTI) investigates brain tissue microstructure in vivo. In multiple sclerosis (MS) Wallerian degeneration of axons traversing focal lesions is a potential mechanism of damage in normal-appearing white matter. In vivo evidence for this hypothesis is limited. The present study investigated the relationship between DTI-derived indices in the normal-appearing corpus callosum (CC) and the lesion loads (LLs) in connected cerebral regions. DTI was performed in 39 MS patients and in 21 age-matched controls. Fractional anisotropy (FA) and mean diffusivity (MD) were estimated in the genu, body and splenium of CC. Patients showed lower FA and higher MD in the CC than controls and both correlated with the total LL (r = -0.56 and r = 0.54, p < 0.0001). The LL of individual cerebral lobes correlated with both FA and MD in the corresponding callosal regions, with the body showing the strongest correlations with frontal and parietal LL (p < 0.0001). The strong correlations between DTI indices in the CC and the extent of lesions in connected brain regions support the hypothesis that Wallerian degeneration of axons transected by remote, but connected focal lesions, is an important pathogenic mechanism of damage in MS.