This study was conducted to investigate the genetic diversity and the relationship between 2 populations including a total 10 genotypes of Berberis crataegina collected from the province of Kayseri in Turkey and 22 genotypes of Berberis sphaerocarpa Kar. & Kit collected from Kyrgyzstan using Inter Simple Sequence Repeat (ISSR) Markers. Twenty ISSR primers used to characterize 32 barberry genotypes and revealed 150 bands of which 111 bands were polymorphic. Band size and the number of bands ranged from 190 to 1400bp and 5 to 14. The average of alleles produced from 20 (ISSR) primers was 7.5. Three primers (TAA)8, (CA)6AC, and (CAC)6 did not show any results whereas, 2 primers (AG)7YC and (TCC)5RY were found to be monomorphic. On the other hand, the rest of primers were polymorphic with polymorphism percentages varied from 28.6 to 100%, with an average of 74%. This study provide that using (ISSR) molecular markers is a reliable method for separating types within a specific genus, and a valuable evidence for decision making in choosing markers for future works, characterization of germplasm, and for contributing of developing Barberry improvement programs.