Almond (Prunus dulcis) displays gametophytic self-incompatibility. In the work reported here, we cloned two novel S-RNase genes from almond cultivar Ferragnes (genotype S1S3) using PCR. The S1-RNase gene has the same coding region as the Sb gene cloned from almond cultivated in the USA; however, their introns are different in sequence. S1 was cloned and sequenced from six different cultivars originating in Europe. The full-length of the S3-RNase gene was cloned using two primers corresponding to the start and stop codons contexts. Two introns are present in the S3 gene, unique among the S-RNase genes. Sequence-specific PCR was performed to confirm that the two cloned genes co-segregate with the S-locus using progenies of a controlled cross between Tuono (S1Sf) and Ferragnes (S1S3). Based on the structural differences of S- and S-like RNase genes, we discuss the evolutionary relationship between the two groups of RNase genes.