In this study, S-allele diversity of eight wild and two commercial species of the Cerasus subgenus in Iran was investigated using two primer pairs. A high level of S-allele polymorphism was detected among and within the species evaluated. Furthermore, most of wild species showed 2–4 alleles based on S-allele primers and may be considered as tetraploid. Sweet cherry cultivars, Siah-Mashhad, Siah-Shabestar, Takdaneh-Mashhad, Siah-Daneshkadeh and Protiva showed S3S12, S3S12, S3S12, S3S5 and S3S4 combinations, respectively, allele S3 showing the highest frequency. Three Iranian sweet cherry cultivars had the same allelic combination (S3S12) that the same ancestor in genealogy of these cultivars may explain the loss of diversity observed at the S-locus. Wild cherry (mazzard) accessions showed wide range of alleles such as S1, S2, S7, S14 and S20 and unknown alleles, while sour cherries showed S6, S9, S13 and S27 alleles. In conclusion, the conservation of these highly diverse native species of Iranian wild Cerasus germplasm is recommended for future breeding activity.