Self-incompatibility (SI) to self-compatibility (SC) transition is one of the most frequent and prevalent evolutionary shifts in flowering plants. Prunus L. (Rosaceae) is a genus of over 200 species most of which exhibit a Gametophytic SI system. Peach [Prunus persica (L.) Batsch; 2n = 16] is one of the few exceptions in the genus known to be a fully self-compatible species. However, the evolutionary process of the complete and irreversible loss of SI in peach is not well understood and, in order to fill that gap, in this study 24 peach accessions were analyzed. Pollen tube growth was controlled in self-pollinated flowers to verify their self-compatible phenotypes. The linkage disequilibrium association between alleles at the S-locus and linked markers at the end of the sixth linkage group was not significant (P > 0.05), except with the closest markers suggesting the absence of a signature of negative frequency dependent selection at the S-locus. Analysis of SFB1 and SFB2 protein sequences allowed identifying the absence of some variable and hypervariable domains and the presence of additional α-helices at the C-termini. Molecular and evolutionary analysis of SFB nucleotide sequences showed a signature of purifying selection in SFB2, while the SFB1 seemed to evolve neutrally. Thus, our results show that the SFB2 allele diversified after P. persica and P. dulcis (almond) divergence, a period which is characterized by an important bottleneck, while SFB1 diversified at a transition time between the bottleneck and population expansion.