Gametophytic self-incompatibility (GSI) is an outcrossing mechanism in flowering plants that is genetically controlled by 2 separate genes located at the highly polymorphic S-locus, termed S-haplotype. This study characterizes a pollen part mutant of the S1-haplotype present in sour cherry (Rosaceae, Prunus cerasus L.) that contributes to the loss of GSI. Inheritance of S-haplotypes from reciprocal interspecific crosses between the self-compatible sour cherry cultivar Újfehértói Fürtös carrying the mutated S1-haplotype (S1' S4SdSnull) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type S1-haplotype revealed that the mutated S1-haplotype confers unilateral incompatibility with a functional pistil component and a nonfunctional pollen component. The altered sour cherry S1-haplotype pollen part mutant, termed S1', contains a 615-bp Ds-like element within the S1-haplotype-specific F-box protein gene (SFB1'). This insertion generates a premature in-frame stop codon that would result in a putative truncated SFBt containing only 75 of the 375 amino acids present in the wild-type SFB1. S1 along with 2 other previously characterized Prunus S-haplotype mutants, Sf1 and S6m, illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of GSI.