Pomegranate is an important horticultural crop in semitropical regions of the world. Cultivation of pomegranate, as income generating fruit to the farmers and export commodity, is also considered in different areas of Iran . Because of widely distribution of native germplasms across the country, pomegranate has enormous genetic variation for different agronomic and quality traits. Given the importance of this diversity, it is important to develop and utilize tools to evaluate genetic diversity among local genotypes to promote conservation and sustainable utilization of this crop. In this study, due to the polymorphic nature of SSRs, combined with their ease of analysis, microsatellite markers were chosen for evaluation of genetic variation among local populations of pomegranate. To design microsatellite primers, two enriched genomic libraries with dinucleotide AG and trinucleotide ATG microsatellite motifs were constructed according enrichment method using magnetic beads. Positives clones have been detected by a simple PCR method contained SSR motifs. Among 43 clones which sequenced with high quality, 32 clones were found containing expected microsatellite repeats (74.4%). A total of 25 SSR primer pairs were designed from flanking regions of the repeats of these positive sequences and their efficiency was assessed among 20 local pomegranate germplasms including 11 commercial cultivars, seven wild and two ornamental genotypes. All primers amplified microsatellite loci in the selected genotypes; 12 of them showed only monomorphic bands, 11 primers ampdifferent clusters. In
