The anti-CD20 antibody ocrelizumab, approved for treatment of multiple sclerosis, leads to rapid elimination of B cells from the blood. The extent of B cell depletion and kinetics of their recovery in different immune compartments is largely unknown. Here, we studied how anti-CD20 treatment influences B cells in bone marrow, blood, lymph nodes, and spleen in models of experimental autoimmune encephalomyelitis (EAE). Anti-CD20 reduced mature B cells in all compartments examined, although a subpopulation of antigen-experienced B cells persisted in splenic follicles. Upon treatment cessation, CD20⁺ B cells simultaneously repopulated in bone marrow and spleen before their reappearance in blood. In EAE induced by native myelin oligodendrocyte glycoprotein (MOG), a model in which B cells are activated, B cell recovery was characterized by expansion of mature, differentiated cells containing a high frequency of myelin-reactive B cells with restricted B cell receptor gene diversity. Those B cells served as efficient antigen-presenting cells (APCs) for activation of myelin-specific T cells. In MOG peptide-induced EAE, a purely T cell-mediated model that does not require B cells, in contrast, reconstituting B cells exhibited a naive phenotype without efficient APC capacity. Our results demonstrate that distinct subpopulations of B cells differ in their sensitivity to anti-CD20 treatment and suggest that differentiated B cells persisting in secondary lymphoid organs contribute to the recovering B cell pool.