The two main objectives of this research were to identify new S-RNase alleles in Iranian almond cultivars and to characterize naturally occurring mutations in these alleles that may cause self-compatibility. We investigated S genotypes of 22 Iranian almond cultivars using stylar RNase electrophoresis, PCR and DNA sequencing. We report six previously unidentified P. dulcis S-RNase alleles (S 45 , S 46 , S 47 , S 48 , S 49 and S 50 ). Four of 12 tested S-RNases were found to be non-functional in vitro: S 49 , S 50 , S 24 /S na and S 25 /S 47 . Detected point mutations in the C3 coding region of S 49 - and S 50 -RNase, leading to the replacement of a highly conserved cysteine and histidine residues, are with the highest probability the reason of these S-RNases inactivity. Results also suggested that ten Iranian almond cultivars display unique S genotype. All presented data confirm Iranian cultivars as valuable almond sources which are of interest to almond breeding and conservation programs.