Summary Chinese pear (Pyrus spp.) exhibits gametophytic self-incompatibility (GSI), as do other fruit species in the family Rosaceae. This work determined S-locus diversity in 20 native Chinese pear cultivars or wild accessions using polymerase chain reaction (PCR) and pollination experiments. After cloning and sequencing the PCR products, the S-genotypes of all 20 pear cultivars or wild accessions were determined. Subsequent sequence analysis showed that P. sinkiangensis Yü ‘Aolian’ (SpS32), P. phaeocarpa Rehd. ‘Diaodan’ (SdSe), P. xerophila Yü ‘Shageda’ (S36Sd) and ‘Xingyeli’ (S22Sc), originating in China, shared some S-RNase genes with P. communis, providing evidence that oriental and occidental Pyrus species may share the same pool of alleles at the S-locus. Two novel S-RNase genes were also discovered in P. ussuriensis ‘Maili’ and ‘Neimenggushanli’, and deposited as S40 and S41, under the accession numbers DQ903313 and DQ988687, respectively. Their deduced amino acid sequences showed high similarity to S11-RNase (100% ) and S6-RNase (94.4%) between the C1 and C3 exons in Malus. The high similarity scores between S-RNases in Pyrus and Malus indicate that the existence of S-RNases predated speciation between Pyrus and Malus.