A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S 2, very similar to S 2 -RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S 2 also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S 1 S 5 and S 2 S 4, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S 2-specific primers, TAC clones containing both AhSLF-S 2 and its homologs were subsequently identified (S 2 TAC, S 5 TACa, S 4 TAC, and S 1 TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S 2, -S 1, -S 4 and -S 5) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S 2 were identified. In total, three F-box genes (AhSLF-S 2, -S 2 A and -S 2 C) in S 2 TAC (51 kb), three (AhSLF-S 4, -S 4 A and -S 4 D) in S 4 TAC (75 kb), two (AhSLF-S 5 and -S 5 A) in S 5 TACa (55 kb), and two (AhSLF-S 1 and -S 1 E) in S 1 TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S 2 C, -S 4 D and -S 1 E) were specifically expressed in pollen, similar to AhSLF-S 2, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.