© 2015 The Japanese Society for Horticultural Science (JSHS), All rights reserved.Flowering plants have developed a genetically determined self-incompatibility (SI) system to maintain genetic diversity within a species. The Solanaceae, the Rosaceae, and the Plantaginaceae have the S-RNase-based gametophytic SI (GSI) system, which uses S-RNase and F-box proteins as the pistil S and pollen S determinants, respectively. SI is associated with culture and breeding difficulties in rosaceous fruit trees, such as apple, pear, and stone fruit species; therefore, researchers in the pomology field have long studied the mechanism and genetics of SI in order to obtain clues to overcome these difficulties. Here, we investigated the evolutionary paths of the S-RNase genes by tracking their duplication patterns. Phylogenetic analysis and estimation of proxy ages for the establishment of S-RNase and its homologs in several rosaceous species showed that the divergence of S-RNase in the subtribe Malinae and the genus Prunus predated the gene in most recent common ancestors of Rosaceae species. Furthermore, the duplicated S-RNase-like genes were accompanied by duplicated pollen S-like F-box genes, suggesting segmental duplications of the S locus. Analysis of the expression patterns and evolutionary speeds of duplicated S-RNase-like genes in Prunus suggested that these genes have lost the SI recognition function, resulting in a single S locus. Furthermore, the S loci in the current Rosaceae species might have evolved independently from the duplicated S loci, which could explain the presence of genus-specific SI recognition mechanisms in the Rosaceae. The results of the present study should be valuable for the future development of artificial SI control and for self-compatible breeding in rosaceous horticultural plant species.