Pomegranate (Punica granatum L.) is a perennial fruit tree native to Iran and the Himalayan Mountains in northern India. In this study, genotyping-by-sequencing (GBS) approach was used for calling single nucleotide polymorphisms (SNPs) as well as genetic variation analysis in a collection of 214 Iranian accessions from different regions. All these individuals were grouped as commercial, wild, base and Ornamental-Pharmaceutical ones. A total of 281514 variants (259,268 SNPs, 11,311 insertions and 10,935 deletions) were identified. The average values of expected heterozygosity (He) and observed heterozygosity (Ho) were 0.22 and 0.26, respectively. The commercial and base groups showed the highest allelic richness. Harmonized results of genetic diversity, allelic richness and genetic differentiation strengthens the hypothesis that most current commercial trees have originated directly from wild genotypes through selection and introduction process rather than classic parental breeding or inbreeding programs. The structure and principal component analyses (PCA) clustered all accessions into three main groups with no special relationship with their geographical origin. For the first time, the LD pattern was investigated in the assayed population, and a partly rapid LD decay with a short range (∼250bp) for all pseudo chromosomes was revealed. This study provides new evidence for research areas in future marker-assisted selective approaches and genome-wide association analyses in pomegranate trees.