S weet cherry (Prunus avium L.) is a species that is characterized by gametophytic self-incompatibility (GSI), which determines the fruit set and, consequently, the tree fruit load. In this study, S-alleles have been identified for nine sweet cherry cultivars in north-western Tunisia. We genotyped 9 cultivars (five introduced unknown cultivars, three international ones and one local ‘Bouargoub’) and we found 2 new Tunisian sweet cherry S-haplotypes (S2 and S10) using polymerase chain reaction (PCR) based on length polymorphism detection of S-RNase and SFB genes. Most introduced sweet cherry cultivars are self-incompatible and cross pollination is necessary to ensure the fruit set. The local cultivar has long been known for gametophytic self-compatible based on the fruit set rate of our research. However, the belonging of S-haplotype (S2, S10) of this cultivar to an allegedly incompatible cultivar group XLII (from the literature) is surprising and emphasizes a hypothesis that the self-compatibility of this last cultivar is unrelated to S-locus. This result is original and can be used in breeding programs. The haplotypes S2 and S10 was less frequent, whereas S3 was the most frequent (44.44%) in the nine studied cultivars.