A new self-incompatibility gene was isolated and identified from Pyrus bretschneideri cultivars of Yingzhiqing and Daaoao via PCR amplification, DNA sequence analysis and cross pollination tests. DNA sequence analysis revealed that the isolated fragment displayed a high homology with S1 ～S11-allele, and the identity to S1～S11-allele at the deduced amino level ranged from 56% to 72%; the high degree of variances in the hypervariable (HV) region resulted from the presence of substitution, deletion and insertion of 9 to 15 amino acids. The new S-allele was named S12-Rnase and its accession number was AY250987 in GeneBank. The sizes of HV region, intron, signal peptide, C1 region, C2 region were 39 AA, 341 bp, 27 AA, 11 AA and 10 AA, respectively. The cross pollination tests were carried out using Pyrus pyrifolia cultivars that contained S1～S9-Rnase genes as female parents, and the cultivars of Daaoao and Yingzhiqing as male parents, respectively. The results showed that all of P.pyrifolia cultivars were compatible with Daaoao and Yingzhiqing, whereas the cross pollination between Daaoao and Yingzhiqing were incompatible, further confirming that the DNA fragment isolated was a new S_gene.