Purpose: To systematically investigate structural and functional alterations of the thalamus and its subregions through a multimodal magnetic resonance (MR) imaging technique and examine its clinical relevance in multiple sclerosis (MS) and neuromyelitis optica (NMO). Materials and Methods: The institutional review board approved this study, and written informed consent was obtained from each participant. Thirty-seven patients with MS, 39 patients with NMO, and 40 healthy control subjects were recruited. Six MR imaging measurements were obtained for each participant and compared between groups in the thalamus and its seven subregions, including gray matter (GM) volume, fractional anisotropy, mean diffusivity, amplitude of low-frequency fluctuation, cross-correlation coefficient of spontaneous low frequency, and weighted functional connectivity strength. Partial correlation was used to estimate the MR imaging-clinical relationships. Results: Both MS and NMO exhibited widespread GM atrophy (GM volume in MS, 0.244; NMO, 0.297; and control subjects, 0.329; P , .001) and diffusion abnormalities (fractional anisotropy in MS, 0.293; NMO, 0.323; and control subjects, 0.355; P , .001) in the whole thalamus and several subregions, while MS showed more severe changes than NMO. Decreased cross-correlation coefficient of spontaneous low-frequency and weighted functional connectivity strength was observed in several thalamus subregions in MS (P , .05), but no significant functional abnormalities were identified in NMO. GM volume, fractional anisotropy, and mean diffusivity, not functional changes of the thalamus and thalamic subregions, correlated with the patients' clinical variables and exhibited high discriminative power in distinguishing the three groups. Conclusion: Similar patterns of thalamic structural alteration were identified in MS and NMO, but MS showed more severe pathologic changes. The thalamus is a key node for functional disconnection in MS but not in NMO.