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Self-incompatibility (SI) has been studied extensively at the molecular level in many members of the Solanaceae, Rosaceae, and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility (GSI). In this study, we analysed 66 new accessions of Chinese cherry (Prunus pseudocerasus) collected in Shandong, Henan, Anhui, Jiangsu, Zhejiang, and Sichuan Provinces. Their S-genotypes were identified using PCR with two conserved or four allelespecific primers. No novel S-alleles were found in these accessions. The frequencies and distributions of the S-alleles were significantly unequal.The frequencies of S1 and S8 were the highest, and those of S4 and S6 were the lowest.The unequal frequencies and distributions of S-alleles could be associated with different climates and/or habitats, or caused by other factors such as natural selection, which would accelerate the evolution of S-alleles. Understanding the frequency and distribution of S-alleles would help to develop informed orchard management strategies for Chinese cherry cultivars.