Scabies occurs in human due to the burrowing ectoparasite Sarcoptes scabiei var. hominis resulting in intense itching and inflammation, and manifesting as a skin allergy. Limited information is available about the genetic diversity of S. scabiei in human. In this study, we characterized S. scabiei var. hominis using nuclear marker ITS-2, mitochondrial marker 16S and microsatellite markers. To examine the extent of the genetic variation, individual mite gDNA was first amplified using ITS-2, 16S and microsatellite primers by PCR, later amplicons were sequenced directly and analysed by MEGA 7. Sequence analysis of ITS2 showed no host segregation and geographical isolation, whereas 16S indicated presence of both hosts adapted and geographically segregated populations of S. scabiei. Results of microsatellites revealed polymorphism as allelic variability between and within the populations studied. The different varieties of Sarcoptes mites belong to different host species and geographic regions recommended that Sarcoptes mites are genetically isolated. This is the first report on the molecular characterization of S. scabiei var. hominis from Pakistan. Additionally, genetic studies including a greater number of specimens are required to comprehend the molecular epidemiology of Sarcoptes mite in Pakistan.