Chloroplast DNA has been used extensively to analyze plant phylogenies at different taxonomic levels because of its size, organization and sequence conservation. In the present research, two chloroplastic regions, petA–psaJ, trnC–trnD and four DNA barcodes (trnH–psbA, ITS, rbcL, matK), were used to introduce suitable regions for the assessment of genetic diversity among P. granatum L. genotypes. Analysis of psbE–petL in petA–psaJ region revealed 1,300 nucleotides with 4.29 % genetic diversity among genotypes, while trnC–petN in trnC–trnD region showed 1.8 % genetic diversity. Therefore, despite the results obtained from the study of other plants, the trnC–trnD region had a low potential for the evaluation of diversity among pomegranate genotypes. Analysis of DNA barcodes in pomegranate showed that trnH–psbA (genetic diversity 2.91 %) provides the highest intra-species variation, followed by ITS (genetic diversity 0.44 %). Eighteen genotypes from different geographical origins of Iran were used to investigate psbE–petL and trnH–psbA potential as novel barcodes to determine genetic polymorphism and characterize pomegranate genotypes. The results suggested that two regions, psbE–petL and trnH–psbA, were more suitable for determining intra-species relationships of pomegranate.