Pediatric-onset MS (POMS) is characterized by a high frequency of brainstem lesions early in the disease. It is unknown whether normal-appearing tissue in this region is involved at this early time point. Using diffusion tensor imaging (DTI) at 3T, we evaluated fractional anisotropy (FA) changes in midbrain substructures in POMS patients and age- and sex-matched healthy controls. Mean FA of midbrain was significantly reduced in the MS group, a difference that remained significant even after removing any focal midbrain lesions. Our results indicate a widespread disruption in the midbrain that exceeds lesional tissue disruption alone.