In the current study, the feasibility and reproducibility of in vivo diffusion tensor imaging (DTI) of the spinal cord in normal mice are illustrated followed by its application to mice with experimental allergic encephalomyelitis (EAE) to detect and differentiate axon and myelin damage. Axial diffusivity, describing water movement along the axonal fiber tract, in all regions of spinal cord white matter from EAE-affected C57BL/6 mice was significantly decreased compared to normal mice, whereas there was no statistically significant change in radial diffusivity, describing water movement across the fiber tract. Furthermore, a direct comparison between DTI and histology from a single mouse demonstrated a decrease in axial diffusivity that was supported by widespread staining of antibody against β-amyloid precursor protein. Regionally elevated radial diffusivity corresponded with locally diminished Luxol fast blue staining in the same tissue from the EAE mouse cord. Our findings suggest that axonal damage is more widespread than myelin damage in the spinal cord white matter of mice with EAE and that in vivo DTI may provide a sensitive and specific measure of white matter injury.