Abstarct: In different parts of Iran, Barberry is used for food and medicine. Seedless Barberry, as an economic crop has a major role in the agricultural economy of the southern region of Khorasan. In recent years, the use of ITS sequences of rDNA has been increased to identify plant species as DNA barcoding. In the present study, the main objective is evaluation efficiency of ITS sequence for DNA barcoding of 19 collected barberry genotypes where they were collected from a barberry collection in Mashhad Food Science and Technology Research Institute and genotypes collected from Esfarayen and Shahroud, Iran. DNA samples were amplified by ITS2 primers and then DNA fragment separated from agarose gel and send to SinaClon Co for sequencing. The phylogenetic analysis of barberry genotypes was performed using MEGA X software by the UPGMA method on the baxse of ITS DNA sequencing. ITS sequences were registered in the NCBI databaxse and the identity of sequences was determined with other related species by blasting analysis. Dendrogram of cluster analysis was done baxsed on sequence similarity with other species in the NCBI databaxse. The results showed that the genetic pair_wise distance, between the studied samples, varied from 0.122 to 0.154. In the cluster analysis, Japanese barberry (JA) was placed in a separate branch from 18 Iranian barberry genotypes. In Blast analysis, 93 DNA sequences of different species of Berberis had more than 90% identity with the ITS sequences of 19 Iranian barberry genotypes in the NCBI databaxse. The most similarity between the ITS2 sequences of ornamental barberry with similar species (B. thumbergii) on the NCBI site was only 91%. In cluster analysis baxsed on sequence on the NCBI site, the R9N3 genotype was separated from the studied barberry genotypes and the other genotypes were divided into two groups. The first group included Esfarayen (ES) genotypes, expect ES1, and Sh1, and R14N2 that most of these genotypes were belong to Berberis crataegina. The second group, including ES1, Sh2-5, R12N1, R8N3, R2N1, R5N1, and R11N3 genotypes, along with other NCBI databaxse genotypes, were classified as B. integerrima. The seedless barberry genotype was also placed in the B. integerrima species class. Due to the high similarity of the DNA sequences, only three pairs of primers were designated for R14N2, R9N3, and B.th genotypes. The DNA of 19 barberry genotypes was amplified by three specific primers. The R14N2 primer was specifically able to reproduce the R14N2 genotype, but the R9N3 and B.th primers did not have a specific band.