Abstract: Pear is a typical fruit tree of gametophytic self-incompatibility. Identifying the S-allele genotype of different pears is an important scientific foundation for arranging varieties for pollination and cross breeding. It also establishes the foundation for inheriting and improving pear cultivars. On the basis of the reported primary structure of pear S1-S9, the primers PF and PR were designed. With the primers and leaf genomic DNA of cultivars Pyrus bretschneideri ‘Eli’ and Pyrus pyrifolia ‘Hakataao’, we us-ed PCR-RFLP,cloning, sequencing and bioinformatic analysis to isolate and identify the two S-alleles of amplification segments with similar size. One of the S-alleles was newly identified and named S34-allele. S-genotypes of the two cultivars were identified as Pyrus bretschneideri ‘Eli’ S13S34 and Pyrus pyrifoli a Hakataao’ S22S34.