Abstract Chinese cherry [Cerasus pseudocerasus (Lindl.) G. Don] is a fruit tree species within the family Rosaceae, with high economic and ornamental values. To verify the ploidy level of this species and compare the karyotypes with relative species, the chromosome number and karyotype characterization of representative wild and cultivated Chinese cherry samples from sixteen natural populations in four Provinces in China, and four Cerasus relative species were investigated. No aneuploids were found in all samples studied and no diploids or triploids were observed among Chinese cherry by root tips from both seedlings and softwood cutting. The chromosomes were quite small in size and mainly composed by median-centromere (m) and submedian-centromere (sm) chromosomes. Fourteen wild and cultivated Chinese cherry samples were all tetraploid with main karyotype formula of 2n = 4x = 32 = 28m + 4sm, samples from Yingjing County as 2n = 5x = 40 and Fengxian County as 2n = 6x = 48, respectively. The ratio between the longest and shortest chromosome ranged from 2.05 to 2.32. All sixteen Chinese cherry samples had karyotypes of “2B” type. Cerasus avium (L.) Moench, C. campanulata (Maxim.) Yu et Li, C. serrulata G. Don var. lannesiana (Carr.), and C. tomentosa (Thunb.) Wall. were all diploid with 2n = 2x = 16, whose karyotypes were “2A”, “1A”, “2B”, and “1B” type, respectively. By comparison of the karyotype formula and characterization, we indicated that C. pseudocerasus showed close relationship with C. serrulata var. lannesiana rather than C. avium. The present study provided powerful important references for the confirmed ploidy level and the possible genome composition of C. pseudocerasus.