Self-incompatibility (SI) is a complex mechanism that prevents plants from self-fertilizing to preserve and promote genetic variability. The angiosperm species have developed two different SI systems, the sporophytic (SSI) and the gametophytic (GSI) systems. SI is a significant impediment to steady fruit production in fruit tree species of the Rosaceae. In Rosaceae, GSI is genetically regulated via a single locus, named the ‘S-locus’, which includes a minimum of two polymorphic and relatively intercorrelated S genes: a pistil-expressed S-RNase gene and several pollen-expressed SFBB (S-locus F-Box Brothers) or SFB (S haplotype-specific F-box protein). This necessitates the interaction of S-RNases with the male determinants. Although genetic and molecular analyses of S genes have shown that mutations in both pistils and pollen-specific components induce self-compatibility in many species and cultivars, other genes or molecules outside the S-locus can co-participate in the male gamete rejection in GSI. However, we highlight and synthesize the most recent knowledge on different mechanisms of GSI in Rosaceae in this current review.