Alexander disease is a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP). How single-amino-acid changes can lead to cytoskeletal catastrophe and brain degeneration remains poorly understood. In this study, we have analyzed 14 missense mutations located in the GFAP rod domain to investigate how these mutations affect in vitro filament assembly. Whereas the internal rod mutants assembled into filaments that were shorter than those of wild type, the rod end mutants formed structures with one or more of several atypical characteristics, including short filament length, irregular width, roughness of filament surface, and filament aggregation. When transduced into primary astrocytes, GFAP mutants with in vitro assembly defects usually formed cytoplasmic aggregates, which were more resistant to biochemical extraction. The resistance of GFAP to solubilization was also observed in brain tissues of patients with Alexander disease, in which a significant proportion of insoluble GFAP were accumulated in Rosenthal fiber fractions. These findings provide clinically relevant evidence that link GFAP assembly defects to disease pathology at the tissue level and suggest that altered filament assembly and properties as a result of GFAP mutation are critical initiating factors for the pathogenesis of Alexander disease.