Self-incompatibility (SI), which has recurred during the evolution of plants, is one of the most important cross-pollination mating systems. Three S-loci have been reported in Brassicaceae, namely, Arabidopsis lyrata (Al), Brassica (Br), and Leavenworthia alabamica (La) S-loci. Here, through multi-genomic comparative analysis of 20 species, we revealed that the most ancient S-locus was formed prior to the divergence of Brassicaceae lineage I and II. It was retained and inherited by Arabidopsis, as the Al S-locus in Brassicaceae lineage I. Furthermore, we found that the Br S-locus, which has been widely used in the breeding of Brassica crops to generate hybrid seeds, was formed through segmental translocation (ST) in the hexaploid ancestor of Brassica in Brassicaceae lineage II. The Br S-locus was evolved through a ST from one of the triplicated ancestral S-locus paralogs in the Brassica hexaploidy ancestor, while the other two S-locus paralogs were lost. Together with the previous discovery that the La S-locus was formed through a secondary origin in Brassicaceae lineage I, we conclude the monophyletic origin of Al and Br S-loci and clarify the evolutionary route of S-loci in the Brassicaceae family. Our findings will contribute to evolutionary studies and breeding applications of the S-locus in Brassicaceae.