The pomegranate ( Punica granatum L. ) is one of the oldest known fruit species. This plant is native to Iran and perhaps some surrounding areas. Pomegranate cultivars distinction is mainly based on morphological characteristics. As morphological characteristic like husk and aril color are affected by agro-ecosystems, for this ancient and widespread fruit it is likely that the same genotypes have different names in different regions (synonymy and/or homonymy). A precise method for determination and discrimination of the genotypes is requested for breeding and commercialization of Iranian pomegranate cultivars which it can be achieved using molecular markers. Nowadays, different molecular markers have been used to determine the genetic diversity among some pomegranate genotypes. Notwithstanding high morphological diversity among the genotypes, low level of polymorphism is indicated in these methods. In this research sequence-related amplified polymorphism (SRAP) markers which target ORFs as functional regions of pomegranate genome and nuclear ribosomal DNA sequences encoding the 5.8S RNA and the flanking internal transcribed spacers (ITS1 and ITS2) were used to detect the genetic diversity within the genotypes and phylogenetic relationships of pomegranate. SRAPs were used to assess genetic diversity of 63 cultivated, wild and ornamental pomegranate genotypes from five different geographical regions of Iran. A total of 250 fragments were amplified using 13 primer combinations (PCs), among which 133 bands (53%) were polymorphic. Average PIC value was 0.28 over all PCs. The genetic distance among genotypes ranged from 0.10 to 0.37 with an average of 0.24. Cluster analysis using the Neighbor-Joining (NJ) method suggested that there are close relationships between ornamental and some wild genotypes. Although the AMOVA results had shown the significant differences in the genetic diversity among the regions ( P value = 0.0048), the genetic variation was mainly caused by the variation of intra regions. The results showed low genetic differentiation ( Fst = 0.025) and high gene flow ( Nm =2.28) among regions. ITS region was also used to evaluate the genetic diversity and phylogenetic relationships of 20 pomegranate genotypes. These data exhibited the presence of polymorphism among pomegranate genotypes. Variation among genotypes demonstrated both in the length and sequence content of ITS region. The size of the spacer’s sequences ranged from 246 to 250 bases for ITS2 and 206 bases for ITS1. ITS2 seems to be relatively more informative than ITS1. Cluster analysis using the maximum parsimony method grouped the genotypes into three clusters. Also predicted conformation of the secondary structure of ITS2 transcripts and thermodynamic stability subjected to detailed iection in comparative analysis among genotypes. Five distinct conformations constructed from ITS2 region of pomegranate genotypes. This method was able to set apart some genotypes as supplement of cluster analysis method. Cluster analysis of ITS2 sequences confirmed previous studies which suggested that Punica genus is actually derived members of the Lythraceae family. In this family Punica genus stand beside Capuronia, Galphinia and Laofensia which is in agreement with previous studies. Keywords: Pomegranate, Genetic diversity, SRAP, Phylogenetic relationships, ITS sequence