Abstract The human thymus has been shown to host B cells, which have been implicated in presentation of autoantigens for negative selection of T cell progenitors. Although these Ags are thought to be taken up through their surface Igs, data on thymic Ig gene repertoires are limited and reactivity to autoantigens has not been demonstrated. We therefore studied the Ig gene repertoires and reactivity to autoantigens of single-sorted B cells from pediatric thymus, and compared these with mature B cells from fetal and pediatric bone marrow. Nearly all B cells in thymus were mature and displayed an Ig gene repertoire that was similar to pediatric bone marrow. Fetal mature B cells predominantly used proximal V, D, and J genes, and their Abs were highly reactive to dsDNA. In contrast, thymic B cells were enriched for autoreactive clones that showed increased specificity to peptide autoantigens. Thus, most B cells in the thymus are resident rather than developing, and are enriched for autoantigen binding. These features support current models for a role of thymic B cells in presentation of autoantigens to developing T cells during negative selection.