© 2022, The Author(s), under exclusive licence to Springer Nature B.V.Pistachio (Pistacia vera L.) is the only cultivated species in Pistacia genus and it is one of the most important species of hard-shelled fruits. Pistachio cultivars have significant level of variation in their phenotypic appearance and productivity. Understanding the genetic diversity between pistachio cultivars could facilitate breeding programs. Simple sequence repeat (SSR) markers are powerful tools in genetic diversity and germplasm collection studies. However, published information about the characterization of large scale pistachio cultivar germplasm with adequate number of SSR markers is limited. In this study, sixty-six pistachio cultivars and genotypes originated from six different countries were characterized and fingerprinted by 74 genomic and 18 genic SSR markers. SSR analysis identified 576 alleles for all 66 cultivars and genotypes. The number of alleles per locus ranged from 2 to 20 (CUPOhBa1592) alleles with a mean value of six alleles per locus. The polymorphism information content (PIC) values ranged from 0.07 (CUPVEST2939) to 0.87 (CUPSiOh2460) with a mean PIC value of 0.58. The pistachio cultivars and genotypes were divided into five clusters according to Structure and UPGMA (Unweighted Pair Group Method with Arithmetic Average) analysis. Total of 61 cultivar specific alleles were detected in 34 cultivars, among them three primers (CUPOhBa1592, CUPBaPa1606 and CUPOhBa2127) produced more than four cultivar-specific loci therefore very promising for cultivar identification, fingerprinting and breeding studies in pistachio.