To finding out the genetic structure of Iranian native horses, as animal blood samples were obtained from diverse areas of Iran, in addition, 114 sequences of equine D-loop region were obtained from GenBank. Total DNA of the samples were extracted through salting out procedure and utilized as template for amplification and sequencing of the D-loop region of mtDNA. Sequence analysis of the 247-bp D-loop region of mtDNA in all samples revealed a total of 44 haplotypes with 39 polymorphic sites. Six haplogroups (A–F) were identified in the phylogenic tree drowned for these haplotypes and sequences from GenBank. Total nucleotide diversity was obtained with in the range of 0.028 ± 0.009 in Iranian native horses. Fixation index values, employing Kimura-2 parameter method ranged from -0.001 to 0.172. Analysis of Molecular Variance (AMOVA) identified that some of these populations, especially Sistani horses, bear a genetic similarity with other populations and also with sequences from the GenBank (P > 0.05). Arab and Turkman horse populations displayed non-significant difference (P > 0.05), which may reflect the uncontrolled crossing between these two breeds. Caspian and Kurd horse populations showed significant differences with other populations and sequences from the GenBank (P > 0.05). The results of the current study indicate a high genetic diversity and as well as genetic similarity in some populations. According to the dateained results it is necessary to control the crosses between the animals populations and the production of progenies in Iranian native horses.