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Objective Iran is one of the few countries where the origin of pomegranate (Punica granatum L.) in the world. About half of the pomegranate genotypes are endangered. Despite the similarity between some genotypes in terms of appearance, there are differences between anthocyanins, phytochemicals, antioxidants, etc., of them that are very much affected by the environment (especially stress) and require DNA barcoding. For this reason, more accurate molecular studies, especially DNA identification, are necessary to aid in classification, identification of the required genotype, non-incorrect naming of genotypes, and identification of cultivars. By performing this research, it is possible to reduce the volume of genotypes and eliminate duplicate and similar genotypes in Saveh pomegranate collection to reduce their maintenance and management costs. Also used to select the best plant barcode to identify, differentiate and determine the diversity of genotypes for use in breeding programs. Materials and methods In this study, 58 genotypes in Saveh pomegranate collection were examined by ITS barcode region. After receiving the sequences, first all the sequences blast in the NCBI site for the accuracy of the desired area and after sure of the desired plant area (pomegranate), the quality of the sequences was measured with Chromas software and in addition to deleting the M13 sequence, the beginning and end sequences and poor-quality sequences were deleted. Then, for multiple alignments by clustalW method, bioinformatics analysis was performed using MEGA software. Results The results showed that the success rate of propagation in this area by PCR was 79%. Also, the success rate of sequencing in this area was 74.68%. The GC content of this area was 64.23%. The lowest genetic distance for this region (0.005) was between the genotypes of Malls Tabas with Chatroud Shirin and the Torsh Dorag Rafsanjan with Globland Bafgh and the highest genetic distance (5.314) was between the genotypes of Gol Magasi Taft with Dane Siyah Ardestan, Poost Ghermez Zanjan, Malas Paveh, Peyvndi Ashkzar and Dane Ghermez Zavareh genotypes. The results of phylogenetic tree also showed that wild genotypes of Tamin Khash, Domezeh Bagh Malek Izeh and Dorag Malas Bajestan and Gol Magasi Taft were each in separate groups and other genotypes were in other groups. Conclusions In general, Ardestan, Khash, Gol Magasi Taft, Malas Peyvandi Ashkzar, Zanjan, Paveh, Zavareh and Ravar genotypes had the highest genetic diversity and distance with other genotypes that according to other characteristics of genotypes, they can be used as parents for breeding programs. Also, considering that this region, unlike other regions, carries both paternal and maternal genes and due to the facilitation of reproduction and the success of their sequences, was identified as a suitable region to show genetic diversity between genotypes which can be used in future research.