The evolutionary history of Pistacia genus and the relationships among the species and accessions are controversial and not well understood. This study reports the application of sequence-related amplified polymorphism (SRAP) technique in characterization of 36 pistachio accessions from Iran, Syria, Turkey and United States by screening 30 primer combinations (PCs). Among them, 11 PCs amplified a total of 202 fragments, of which 168 (83%) were polymorphic, with an average of 15.27 fragments for each PC. The cluster analysis revealed species-based grouping and the closest species of Pistacia vera is found to be Pistacia atlantica followed by Pistacia khinjuk. The results of AMOVA analysis demonstrated that most of the total molecular variance was attributable to divergence among the accessions from different species. Different P. vera accessions with wild “Sarakhs” accession clustered in a separate group based on SRAP markers. It can be assumed that the domesticated genotypes of P. vera evolved from “Sarakhs” accession.