Both genes exhibit high levels of polymorphism and sequence diversity, characteristic of plant self-incompatibility systems. In this study, eight S-RNase alleles and five SFB alleles were isolated from five accessions of P. virginiana using PCR. These alleles were novel and have been submitted to GeneBank with the following Accession Numbers: S1-RNase – S8-RNase (JF907557 – JF907564), SFB1 (JF907565), SFB2 (JF907566), and SFB4 (JF907567).The Accession Numbers of SFB7 and SFB8 are the same as S7-RNase and S8-RNase, respectively. These S-RNase and SFB alleles shared the typical structural features of the equivalent alleles in other Prunus species. The identities of the deduced amino acid sequences range from 70.5 – 80.9% among the S-RNase alleles, and from 66.5 – 82.9% among the SFB alleles. The S-RNase and SFB genes were transcribed in opposite directions. The distances between pairs of alleles in these S-haplotypes varied from 325 – 1,789 bp. Sequence comparisons showed that the identities of the deduced amino acid sequences were 99.4% between Pvir-S3 and Pweb-S10, 99.0% between Pvir-S3 and Pd-S64, 97.1% between Pvir-S7 and Pps-S5, 98.2% between Pvir-S4 and Pa-S6, 98.2% between Pvir-S6 and Pspe-S3, 98.4% between Pvir-SFB4 and Pa- SFB6, and 97.4% between Pvir-SFB7 and Pps-SFB5. Moreover, the identity between the intergenic sequences of the S7-haplotype of P. virginian and the S5-haplotype of P. pseudocerasus was 99.7%, with only one nucleotide difference (C →T).These results show that these two S-haplotypes were the closest related lineages and further support the view that alleles at these two S-loci evolved before the divergence of Prunus species.