In this study, eight S-RNase alleles were isolated from two Prunus pseudocerasus and two Prunus speciosa accessions by PCR amplification from genomic DNA. Analysis of the amino acid sequences revealed five novel and three published S-alleles. These S-RNases share typical structural features with S-RNases from other Prunus species exhibiting gametophytic self-incompatibility. The deduced amino acid identities ranged from 60.8 to 75.6% among four S-RNase alleles in Prunus speciosa and ranged from 73 to 81.4% among four S-RNase alleles in Prunus pseudocerasus. The size of the first introns ranged from 197 to 341 bp, and the size of second introns ranged from 81 to 1182 bp. Sequence analysis demonstrated that the deduced amino acid identities, by comparison with other Prunus species, were often higher than those of intraspecific identities. Moreover, exceptionally high identities were found between Pspe-S1 and Pd-S28; between Pspe-S31 and Pm-S6; among Pspe-S51, Pa-S29 and Pweb-S7; and among Pps-S13, Psim-S4 and Ps-Sb, indicating that the S-RNase alleles evolved before Prunus species divergence. Interestingly, the similarities of the first and second introns were also high between the two S-RNase alleles, which range from 83.63 to 100% among the first introns and from 46.13 to 100% among the second introns. These information could not increase our knowledge on the S-alleles of Prunus species, but is available for molecular breeding of fruit trees, to avoid cross-incompatibility.