In Rosaceae species, two gametophytic self-incompatibility (GSI) mechanisms are described, the Prunus self-recognition system and the Maleae (Malus/Pyrus/Sorbus) non-self- recognition system. In both systems the pistil component is a S-RNase gene, but from two distinct phylogenetic lineages. The pollen component, always a F-box gene(s), in the case of Prunus is a single gene, and in Maleae there are multiple genes. Previously, the Rosa S-locus was mapped on chromosome 3, and three putative S-RNase genes were identified in the R. chinensis 'Old Blush' genome. Here, we show that these genes do not belong to the S-locus region. Using R. chinensis and R. multiflora genomes and a phylogenetic approach, we identified the S-RNase gene, that belongs to the Prunus S-lineage. Expression patterns support this gene as being the S-pistil. This gene is here also identified in R. moschata, R. arvensis, and R. minutifolia low coverage genomes, allowing the identification of positively selected amino acid sites, and thus, further supporting this gene as the S-RNase. Furthermore, genotype-phenotype association experiments also support this gene as the S-RNase. For the S-pollen GSI component we find evidence for multiple F-box genes, that show the expected expression pattern, and evidence for diversifying selection at the F-box genes within an S-haplotype. Thus, Rosa has a non-self-recognition system, like in Maleae species, despite the S-pistil gene belonging to the Prunus S-RNase lineage. These findings are discussed in the context of the Rosaceae GSI evolution. Knowledge on the Rosa S-locus has practical implications since genes controlling floral and other ornamental traits are in linkage disequilibrium with the S-locus.