The Chinese pear (Pyrus spp.) exhibits typical gametophytic self-incompatibility (GSI), which inhibits self-crossing and promotes out-crossing, similar to other fruit species in the Rosaceae family. Thus, S-compatible cultivars are required in pear orchards to ensure successful pollination and stable yields. In this study, 84 native Chinese pear accessions were genotyped by allele-specific PCR using one pair allele consensus primers and 29 pairs of S-allele-specific primers that were designed in this study. After cloning and sequencing the PCR products, the S-genotypes of all 84 pear accessions, including wild and cultivated accessions, were determined. The reported 34 S-alleles and a novel S-allele were isolated from these pear accessions. These S-alleles were expressed specifically in the style. Sequence analysis identified that six pear cultivars originated in China shared the same S-RNases with P. communis (Pc). These findings supported the hypothesis that oriental and occidental Pyrus spp. may share the same pool of alleles at the S-locus. A novel S-RNase was isolated in ‘Putiandouli’, ‘Daguoshanli’, ‘Yunhongli 1′, and ‘Dianli’ and deposited as S₆₇-RNase under accession number MT773568. Furthermore, the deduced amino acid sequences exhibited high similarity (99.56%) to S₃₂-RNase in Malus. The high similarity between S-RNase in Pyrus and Malus indicated that the existence of S-RNase could have predated speciation between Pyrus and Malus. Furthermore, S-allele information was rearranged in Asian and European pears to provide information for selecting the best pollinator for widely cultivated pear cultivars in China. This information is useful for pear production, cross-breeding, and understanding the mechanism of the self-incompatibility reaction.