Malus S-RNase genetic diversity was analyzed in Malus x domestica cultivars and compared to European wild apple (Malus sylvestris). Using PCR-based approaches, the S-RNase genotype of 140 M. x domestica cultivars, 196 M. sylvestris trees and 27 M. sylvestris-M. x domestica hybrids was determined. S-RNase allelic richness in M. sylvestris was much higher than in M. x domestica, indicating the negative influence of domestication on S-RNase diversity. Heterogeneity of the S-RNase allelic distribution is much higher in cultivated apple than in wild apple, which shows that breeding leads to strong departure from the expected homogeneity of genes under negative frequency-dependent selection. The majority of the M. x domestica S-alleles has been found in M. sylvestris as well, which points to strong conservation of the S-locus gene structure. Based on the sequence of all different SCAR-fragments, which comprise both the hypervariable PS1 region and the single intron, S-RNase genetic diversity was further explored. It provided some clues to the occurrence of new S-alleles among the multitude of novel S-RNase sequences that have been identified, which were mostly unique for the group of M. sylvestris individuals. The determination of the S-RNase genotypes of old cultivars and M. sylvestris will enable their introduction into new breeding strategies. As M. sylvestris has become an endangered species in Belgium, the knowledge gathered in this study will be an important tool for selecting useful genotypes for a core collection.