Capuli (Prunus serotina subsp. capuli) is a tree species that is widely distributed in the northern Andes. In Prunus, fruit set and productivity appears to be limited by gametophytic self-incompatibility (GSI) which is controlled by the S-Locus. For the first time, this research reveals the molecular structure of the capuli S-RNase (a proxy for S-Locus diversity) and documents how S-Locus diversity influences GSI in the species. To this end, the capuli S-RNase gene was amplified and sequenced in order to design a CAPS (Cleaved Amplified Polymorphic Sequence) marker system that could unequivocally detect S-alleles by targeting the highly polymorphic C2-C3 S-RNase intra-genic region. The devised system proved highly effective. When used to assess S-Locus diversity in 15 P. serotina accessions, it could identify 18 S-alleles; 7 more than when using standard methodologies for the identification of S-alleles in Prunus species. CAPS marker information was subsequently used to formulate experimental crosses between compatible and incompatible individuals (as defined by their S-allelic identity). Crosses between heterozygote individuals with contrasting S-alleles resulted in normal pollen tube formation and growth. In crosses between individuals with exactly similar S-allele identities, pollen tubes often showed morphological alterations and arrested development, but for some (suspected) incompatible crosses, pollen tubes could reach the ovary. The latter indicates the possibility of a genotype-specific breakdown of GSI in the species. Overall, this supports the notion that S-Locus diversity influences the reproductive patterns of Andean capuli and that it should be considered in the design of orchards and the production of basic propagation materials.