Sweet cherries express self-incompatibility, which affects the choice of parents and mating combination in a breeding program. Self-incompatibility is controlled by a multi-allelic single locus (S-locus). Here, the S-alleles were first time studied in 37 local sweet cherry genotypes native to Croatia by using polymerase chain reactions (PCR). Thirteen specific primers designed for the amplification of S1, S2, S3, S4, S5, S6, S7, S9, S10, S12, S13, S14 and S16 alleles were employed. Alleles S3 and S12 were found to have the highest frequencies (39 and 19%, respectively) while S1 and S4 were observed in the lowest frequency (3%) among the examined genotypes. The results obtained, together with information compiled from previous studies, allowed the extension of a total of 10 formerly described cross-incompatibility groups (CIG) determined among the 37 Croatian sweet cherry genotypes. S3S12 was the most frequent allelic combination (49%), which was observed in 18 Croatian sweet cherry genotypes. A combination of consensus and allele-specific PCR amplification was capable enough to identify all the S-alleles in the genotypes studied. The method also provided a rapid and efficient approach for identifying the S-genotypes of sweet cherry genotypes native to Croatia. The results are indicating possible genetic relationships between Croatian and Turkish sweet cherry cultivars as well. © 2012 Elsevier B.V.