Objective: The coding of astigmatid mites based on their morphological and developmental characteristics often leads to uncertainty in the results. The ribosomal internal transcribed spacer (ITS-2) region, being highly conserved in eukaryotes is commonly employed as a barcode for identification of mite species. The present study was an attempt to characterize the gene sequences of astigmatid mites i.e. Sarcoptes scabiei (S. scabiei), Dermatophagoides farinae (D. farinae) using ITS-2 as a genetic marker. Place and Duration of Study: The study was conducted at Department of Dermatology, Military Hospital (MH), Rawalpindi from September 2012 to October 2013. Materials and Methods: In order to characterize relationship of astigmatid mites, the ITS-2 marker was successfully amplified and sequenced. The resulting ITS-2 gene sequences were aligned using Clustal W. MEGA 7 was used to construct phylogenetic tree of the aligned sequence. Results: The phylogenetic tree showed an overall genetic distance of 0.53 indicating close genetic relationship among astigmatid mite species. Pairwise distance was calculated for the ITS-2 gene and low genetic diversity values were observed within S. scabiei and D. farinae that range from 0.003-0.008 and 0.006-0.038 respectively. Conclusion: The study supports the view that the ITS-2 region can be used to identify morphologically difficult astigmatid mites but is not useful in characterization of different species based on the geographical distribution. This study has important implication in our understanding of the epidemiology of S. scabiei and D. farinae and development of control strategies in human transmission.