Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4⁺Foxp3⁺ regulatory T (T_reg) cells^, generated in the thymus or extrathymically by induction of naive CD4⁺Foxp3⁻ T cells. Previous studies suggested that the T-cell receptor repertoires of thymic T_reg cells and induced T_reg cells are biased towards self and non-self antigens, respectively^,,,, but their relative contribution in controlling immunopathology, such as colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved. The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which T_reg cells and other T-cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage^, and favour tolerogenic presentation of antigens to naive CD4⁺ T cells^,, suggesting that intestinal homeostasis depends on microbiota-specific induced T_reg cells^,,,. Here, to identify the origin and antigen-specificity of intestinal T_reg cells, we performed single-cell and high-throughput sequencing of the T-cell receptor repertoires of CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ T cells, and analysed their reactivity against specific commensal species. We show that thymus-derived T_reg cells constitute most T_reg cells in all lymphoid and intestinal organs, including the colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that thymic T_reg cells, and not induced T_reg cells, dominantly mediate tolerance to antigens produced by intestinal commensals.