Japanese apricot (Prunus mume Sieb. et Zucc.) originated in China with more than 500 cultivars. Most cultivars are self-incompatible. It is essential to identify S-genotypes for production and breeding. The S-genotypes of 17 Japanese apricot cultivars native to China were determined using the Prunus S-RNase consensus primer pair, Pru-C2 and PCE-R, which were designed from the second and third conserved regions of Prunus S-RNases, respectively. Eleven S-RNase alleles (S-1 S-2, S-7, S-12, S-14, S-15, S-18, S-20, S-22, S-23 and S-26) and four new S-RNase alleles (S-30, S-31, S-32 and S-33) with GenBank Accession Numbers JN232975, JN232976, JN232977, JN232978 were identified. Furthermore, the S-genotypes of four Japanese apricot cultivars were confirmed by field-testing for cross-pollination. The results of this study enrich the information available on S-genotypes in Japanese apricot and provide a reasonable basis for the appropriate arrangement of pollination trees in orchard practice.