OBJECTIVE: To apply a novel postprocessing voxel-based analysis for diffusion tensor imaging of the cervical spinal cord in multiple sclerosis (MS) in a prospective cross-sectional study. METHODS: Fourteen patients with MS who were within 4 weeks of the onset of cervical myelitis (lesion C1-3) and 11 healthy controls underwent cervical spinal cord diffusion tensor imaging. Cervical spinal cord maps of fractional anisotropy (FA), mean diffusivity, radial diffusivity (RD), and axial diffusivity were registered and compared between patients and controls. Mean FA and RD values from significant thresholded clusters were regressed with clinical scores, after adjusting for cord area and age, to determine associations with physical disability. RESULTS: Cord registrations for subjects were qualitatively assessed (scored out of 5) and those with low scores (1 or 2) were excluded from further analysis. Cord registration was considered good in 11 patients (6 females; mean age = 35.5 years) and 10 controls (6 females; mean age 44 years). Voxel-based comparisons showed patients with MS had lower FA and higher RD at C2-3 levels (left >right mainly in gray matter; p < 0.01, uncorrected). Extracted values of both FA and RD from thresholded clusters were significantly associated with greater disability measured using the Expanded Disability Status Scale and Timed 25-Foot Walk Test in patients with MS. CONCLUSIONS: Mapping diffusion abnormalities within the cervical spinal cord using a novel voxel-based approach can localize clinically relevant pathology.