Summary Many species of Prunus display an S ‐RNase‐based gametophytic self‐incompatibility (SI), controlled by a single highly polymorphic multigene complex termed the S ‐locus. This comprises tightly linked stylar‐ and pollen‐expressed genes that determine the specificity of the SI response. We investigated SI of Prunus tenella , a wild species found in small, isolated populations on the Balkan peninsula, initially by pollination experiments and identifying stylar‐expressed RNase alleles. Nine P. tenella S ‐ RNase alleles ( S 1 – S 9 ) were cloned; their sequence analysis showed a very high ratio of non‐synonymous to synonymous nucleotide substitutions ( K a / K s ) and revealed that S ‐ RNase alleles of P. tenella , unlike those of Prunus dulcis , show positive selection in all regions except the conserved regions and that between C2 and RHV. Remarkably, S 8 ‐RNase, was found to be identical to S 1 ‐RNase from Prunus avium , a species that does not interbreed with P. tenella and, except for just one amino acid, to S 11 of P. dulcis. However, the corresponding introns and S‐RNase – SFB intergenic regions showed considerable differences. Moreover, protein sequences of the pollen‐expressed SFB alleles were not identical, harbouring 12 amino‐acid replacements between those of P. tenella SFB 8 and P. avium SFB 1 . Implications of this finding for hypotheses about the evolution of new S ‐specificities are discussed.