Tetraploid sour cherry (Prunus cerasus) exhibits a genotype-dependent loss of gametophytic self-incompatibility that is caused by the accumulation of non-functional S-haplotypes with disrupted pistil component (stylar-S) and/or pollen component (pollen-S) function. Genetic studies using diverse sour cherry germplasm identified non-functional S-haplotypes for which an equivalent wild-type S-haplotype was present in sweet cherry (Prunus avium), a diploid progenitor of sour cherry. In all cases, the non-functional S-haplotype resulted from mutations affecting the stylar component S-RNase or Prunus pollen component S-haplotype-specific F-box protein (SFB). This study determines the molecular bases of three of these S-haplotypes that confer unilateral incompatibility, two stylar-part mutants (S(6m2) and S(13m)) and one pollen-part mutant (S(13)'). Compared to their wild-type alleles, S(6m2)-RNase has a 1 bp deletion, S(13m) -RNase has a 23 bp deletion and SFB(13)' has a 1 bp substitution that lead to premature stop codons. Transcripts were identified for these three alleles, S(6m2)-RNase, S(13m)-RNase, and SFB(13)', however, these transcripts presumably result in altered proteins with a resulting loss of activity. Our characterization of natural pollen-part and stylar-part mutants in sour cherry along with other natural S-haplotype mutants identified in Prunus supports the view that loss of pollen specificity and stylar rejection evolve independently and are caused by structural alterations affecting the S-haplotype. The prevalence of non-functional S-haplotypes in sour cherry but not in sweet cherry (a diploid) suggests that polyploidization and gene duplication were indirectly responsible for the dysfunction of some S-haplotypes and the emergence of self-compatibility in sour cherry. This resembles the specific mode of evolution in yeast where accelerated evolution occurred to one member of the duplicated gene pair.