Almond is a highly heterozygous species with a high number of S-alleles controlling its gametophytic self-incompatibility system (GSI). In this work, we have analysed 14 Spanish local almond cultivars for S-RNase allele diversity. Five new S-RNase alleles were identified by cloning and sequencing, S31 (804 bp) in ‘Pou de Felanitx’ and ‘Totsol’, S32 (855 bp) in ‘Taiatona’, S33 (1165 bp) in ‘Pou d’Establiments’ and ‘Muel’, S34 (1663 bp) in ‘Pané-Barquets’ and S35 (1658 bp) in ‘Planeta de les Garrigues’. Additionally, seven already known almond alleles could be recognized in the local cultivars studied. The high number of new alleles identified reveals the wide diversity of almond germplasm still existing and requiring characterization, and points to the possibility of new findings by a wider study focusing on other provenances. The almond S-RNases have been compared to those of other Prunus species, showing a high identity and confirming that the S-RNase gene in this genus presents a probable common ancestor.