Histological studies suggest that white matter microstructure varies across different subregions of the corpus callosum (CC). We used mcDESPOT-derived measures to examine myelin content and thickness in vivo in 3 subregions of the CC in healthy controls (HC) and individuals with neuromyelitis optica spectrum disorder (NMOSD). Differences in both myelin content and thickness were observed in different subregions of the CC in HC. Myelin content was decreased in posterior CC in NMOSD relative to HC. mcDESPOT-derived myelin measurements are sensitive to differences in white matter microstructure and can be used to investigate the underlying pathology contributing to demyelinating diseases.