The S-RNase-based gametophytic selfincompatibility (GSI) system, being present in members of the three families Rosaceae, Solanaceae and Plantaginaceae, is one of the most widespread mechanisms that plants have evolved to prevent inbreeding and promote outcrossing. Despite being evolutionarily a highly favorable trait for those species who have perfect flowers, self-incompatibility is an undesired feature for some crops, such as pome and stone fruits, in which fruit set is directly dependent on the success of the fertilization process; for this reason, it has been extensively studied in the last years. It acts through a specific pollen-pistil recognition mediated by female (pistil-expressed) and male (pollen- expressed) proteins, whose genes lay in the single, multigenic and multiallelic S locus. The female S determinant is a stylar ribonuclease (S-RNase), whereas its male counterpart was only recently identified as a single or multiple pollen-specific F-box proteins. In incompatible pollen tubes, the S-RNases act as cytotoxins, causing the degradation of the cellular RNA and likely triggering a programmed cell death (PCD) program. If the pollen is recognized as “nonself”, on the contrary, S-RNases are inhibited, presumably through a specific proteolytic degradation by the ubiquitin-proteasome pathway. Several studies in the last years contributed to unveiling the structure and function of the S locus genes in Rosaceous species. Surprisingly, a key difference seems to exist between the Prunus group and the Pyrinae, such as apple and pear. In the first group the pollen S function is provided by a single S-locus F-box gene, named SFB (for S-haplotype-specific F-Box), that seems to interact in an allele-specific manner with the “self” SRNase to protect it from inhibition; in apple and pear, on the contrary, the pollen S function seems to be exerted by a pool of F-box proteins named SFBB (Slocus F-Box Brothers), each having specificity for one or a few “non-self” S-RNase alleles; each S haplotype contains a number of SFBBs sufficient to recognize all the S-RNase alleles except the “self” one. In this case, contrarily to Prunus, the specific interaction between a F-box protein and the S-RNase leads to its inhibition rather than its protection; nevertheless, the result is the same: only the “self” S-RNase can escape inhibition and remains active to trigger pollen rejection. The study of self-incompatibility in pome and stone fruits, besides its importance for understanding key mechanisms in plant reproduction, has provided along the last years valuable tools of practical interest for growers and breeders: PCR-based S-genotyping assays proved to be a fast and reliable method to determine compatibility groups of varieties, and the molecular characterization of spontaneous and induced mutants facilitated the selection of new self-compatible cultivars in several species.