Bark beetles of Scolytinae subfamily are known as one of the most important pests on horticultural and forest trees, which mainly attack the weak and under environmental tensions trees, and may cause so many damages to the trees. The Mediterranean pine bark beetle ( Orthotomicus erosus ) is one of the most destructive bark beetles of pine trees in Iran. This species can be distinguished morphologically from the other species in most regions by four ending protrusions at the terminal section of its elytra which the second spine is wide with smooth and round edge. The analysis of mitochondrial DNA provides important information about the genetic diversity of insect populations. For molecular identification and genetic diversity analysis of O. erosus , several populations were collected from Isfahan and other regions in Iran and a 488-base pair region of COXI and a 383-base pair region of 16S rDNA were PCR amplified and sequenced. applied. CTAB method was used to extract the samples' DNA. The phylogenetic tree constructed using Neighbor joining and Maximum Parsimony showed a little distance and diversity among the populations. Furthermore, the phylogenetic tree could somehow separate the samples given to their sources. Locating positions of Iranian samples meet the Orthotomicus and does not have monophyletic case with the other congeneric species such as O. caelatus and heterogeneric species such as Xylosandrus germanus . The Molecular clock results showed that the relationship among the analysed populations dates back to less than 0.1 million years ago and it does not pass a long time from their separation from each other. The observed intraspecific genetic distance was completely compatible with the distance among other insects. Interspecific genetic diversity was higher than the intraspific diversity in both genes, and in some cases they overlapped. The minimum and maximum genetic distance for COXI gene and 16S rDNA gene were (0, 0.235) and (00.2, 0.101), respectively. The mismatch distribution analysis for both genes in the tested populations showed the same results, the curves did not show normal distribution, however, it was compatible to population expansions from small populations. The main genetic diversity parameters including haplotypes numbers, haplotypes diversity, nocleotic diversity, polymorphism site numbers and the pair nucleotide diversity mean (MPD) for the both given genes were calculated. The ordering and arrangement results showed 23 and 111 locations in positioning sites of DNA organic bases for COXI , and 16S rDNA . The genetic diversity of 0.03, Tajima -0.819, Fu's -0.801 were calculated for COXI gene, while, the genetic diversity of 0.06, Tajima -1.75, and Fu's -1.817 were calculated for 16S rDNA that shows the recent development of bark beetle populations in Iran, or the natural selection effect on these gene and on the balanced genetic mutation drift process along the development. From the both used mitocondrial gene, 15 haplotypes were related to COXI gene and 14 haplotypes were observed in 16S rDNA . The haplotype diversity related to the COXI gene and the 16S rDNA gene were obtained 1.000 and 0.990, repectively. In conclusion, 16S rDNA gene showed a higher genetic diversity and as a result, is more competent rather than the COXI gene in analysing the genetic diversity levels of these species populations. Keywords: Mediterranean pine bark beetle, Genetic diversity , Phylogeny , COX1 , 16S rDNA , Orthotomicus erosus