Identification of S-genotypes of pear cultivars is useful for allocating suitable pollination trees and pear breeding program. The fluorescently-labeled specific cDNA sequences of S-RNases were obtained by PCR amplification using Cy3-labeled specific primers. The S-RNase cDNA microarray was further improved with which the pear S-genotype was detected and novel S-RNase genes were discovered. The S-genotypes of thirty-five pear cultivars，in total, including ‘Luding Wangpili’, ‘Xingshan 24’ and ‘Midu Baihe’ etc, were identified and seven new P. pyrifolia S-RNase genes were discovered. Then the DNA sequences of the seven new S-RNase genes were identified by DNA cloning and sequencing combined with PCR-RFLP method. Sequence analysis demonstrates that the seven new genes showed typical structural features with specific domains of S-RNase. Evolution analysis indicates that the seven genes blong to the S-RNase group of Maloideae, Rosaceae and the evolutionary relationship among S-RNases is closer between species or genera than intra-species or intra-genus. The seven S-RNases were named PpS53 (Pyruspyrifolia S53), PpS54, PpS55, PpS56, PpS57, PpS58 and PpS59, and have been deposited under the GenBank database with accession numbers being KX581753, KX581754, KX581755, KX581756, KX581757, KX581751 and KX581752, respectively.