Gametophytic self-incompatibility (GSI) is a genetically controlled mechanism to prevent inbreeding and promote out-crossing. Pear is a commercially important fruit tree in China that exhibits GSI. It is necessary to determine the compatibility between cultivars for determination of cross-compatible combination prior to performing pear plantation and breeding programs. Generally, the compatibility between cultivars was examined by field pollination. However, the results acquired by field pollination were vulnerable to environmental impacts and always inaccurate. So cloning the pear S-RNase that controls self-incompatibility is helpful to exactly clarify the compatibility or incompatibility between cultivars. In this study, S29-RNase cDNA was cloned by rapid amplification of cDNA ends (RACE) method in the cultivar `Mili' (S19S29). S29-RNase displayed typical sequence features of pear S-RNases, i.e. five conserved regions and a hypervariable (HV) region, indicating that it behaves as RNase rejecting pollen tube growth with the same S-haplotype. At the amino acid level, S29-RNase showed 32% to 91% similarities with other rosaceous S-RNases. Phylogenetic analysis revealed that rosaceous S-RNases occurred before divergence of species, but after divergence of subfamilies Maloideae and Amygdaloideae. This study will be helpful in pear production and breeding programs.