There are limited numbers of pistachio (Pistacia vera L.) cultivars in the world and their phenotypic appearance and productivity are variable. Understanding such variation would facilitate their use in cultivar breeding programs. Therefore, in this study, 69 pistachio cultivars and genotypes originating from seven countries were characterized by randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), and amplified fragment-length polymorphism (AFLP) markers. The results showed that all three marker systems were able to reveal variability between pistachio cultivars and genotypes. The correlation coefficients for genetic distances were statistically significant among all three molecular marker types. The correlation between RAPD and AFLP data was the highest (r = 0.73) and the value between RAPD and ISSR data was the lowest (r = 0.58). AFLP proved to be the best technique among them. ISSR and AFLP assays were reliable and produced reproducible bands. ISSR was preferred over RAPD, especially when financial investment and technical knowledge are limited. The constructed unweighted pair group method with arithmetic averages (UPGMA) dendrogram obtained from combined data separated the genotypes into two main clusters: one cluster ("Iranian") included genotypes originating from Iran and the second cluster ("Mediterranean") contained most other genotypes. The "Mediterranean" cluster further divided into three subclusters: one ("Siirt") consisted of the cultivars Siirt and Hacireso with a few other selections; the second subcluster ("Turkish") included Turkish cultivars; and the third subcluster contained Syrian, Italian, and the remaining cultivars. The closeness of the clusters was "Iranian" - "Siirt" - "Turkish"/"Syrian." These findings reveal a new understanding in the diffusion of pistachio cultivation from its center of origin, the Iranian-Caspian region, via southeastern Turkey to Syria, the Mediterranean region of Europe, and northern Africa.