Gu, C., Wang, L., Korban, S. S. and Han, Y. 2015. Identification and characterization of S-RNase genes and S-genotypes in Prunus and Malus species. Can. J. Plant Sci. 95: 213-225. Most Rosaceae fruit trees such as Prunus and Malus species exhibit gametophytic self-incompatibility that is genetically controlled by the S-locus. In turn, the S-locus contains at least two tightly-linked S-determinant genes, a pistil S-RNase and a pollen SFB. In this study, S-genotypes of 120 cultivated and wild Prunus accessions (peach) and seven wild Malus accessions (crabapple) have been characterized. Among cultivated Prunus genotypes, four S-RNase alleles, designated S₁, S₂, S₃, and S₄, have been identified, and they share typical structural features of S-RNases from all other self-incompatible Prunus species. Four S-genotypes, S₁S₂, S₁S₃, S₁S₄, and S₂S₂, were identified in peach cultivars, while only one S-genotype S₁S₂ for wild Prunus species. The S₁S₂ genotype is predominant in peach cultivars, accounting for 58.3% of all evaluated accessions. Similarly, four SFB alleles were identified in peach cultivars and wild accessions. However, all the four SFB alleles encode truncated proteins due to a frame-shift mutation, resulting in loss of hyper-variable and/or variable regions. For Malus species, a total of 14 S-RNase alleles are identified, and of those, two alleles encode truncated proteins. Overall, the genetic variation of both S-RNase and SFB genes in peach is significantly lower than that of S-RNase and SFB genes in self-incompatible Malus and/or Prunus species. The relationship between the genetic variation of SFB genes and the diversification of S-RNase genes in Rosaceae is also discussed.