Gametophytic self-incompatibility (GSI) in sweet cherry (Prunus avium, Rosaceae) is controlled by the multi-allelic S-locus. This complex locus contains two linked genes, an S-RNase that controls pistil specificity, and an F-box gene named SFB (S-haplotype-specific F-box protein gene) that is the putative determinant of pollen specificity in Prunus species. The S-locus is considered to be a region of repressed recombination, as recombination has not been observed. Recombination between the pistil-S and pollen-S determinant genes would result in non-functional S-haplotypes and loss of self-incompatibility. With the recent identification of multiple SFB alleles in sweet cherry, along with the existing S-RNase alleles, it is now possible to estimate the linkage distance and quantify the physical distance between the two GSI specificity genes in multiple sweet cherry S-haplotypes. A recombinational analysis between S-RNase and SFB was performed for four sweet cherry S-haplotypes (S-2, S-3, S-4, S-6) using F-1 progeny from reciprocal crosses between 'Emperor Francis' (S-3 S-4) and 'NY 54' (S-2 S-6). For SFB genotyping, allele-specific primer sets were designed from the sequence of the SFB coding region. The S-RNase and SFB genotypes of 511 progeny, representing the outcomes of 1,022 meioses, were determined. All four S-haplotypes individually segregated according to the expected Mendelian ratio of 1:1. The S-RNase and SFB loci were completely linked as no recombinant individuals were identified, thus maintaining the co-evolved allele specificities for the pistil and putative pollen determinant. The physical distance between S-RNase and SFB in six sweet cherry S-haplotypes (S1-S6) was determined using PCR with genomic clones as the template. The relative order and transcriptional orientation of S-RNase and SFB were conserved across the six S-haplotypes. However, the physical distance between these two genes varied widely, ranging from 380 bp to approximately 40 kb. This study represents the first large-scale recombinational analysis of the S-locus region in the Rosaceae, serving as the starting point for future comparative analyses of physical distances, linkage distances, and sequence diversity among Prunus S-haplotypes.