Psoroptic mange is one of the most common contagious skin conditions causes economic losses in livestock. The present study determine the prevalence of Psoroptes spp. in cattle and buffaloes in Sharkia governorate, Egypt and associated risk factors as season, age and sex. Psoroptes isolates of buffaloes were identified morphologically using light and scanning electron microscope and molecularly using PCR and sequencing against the second internal transcribed spacer (ITS2 gene). The current study revealed that prevalence of Psoroptes spp. in cattle and buffaloes was 4.8% and 27.4% respectively. On the other hand, using sequences analysis showed that the isolates collected from buffaloes were Psoroptes natalensis. Morphometric analysis was compatible with previous studies and discerns that there was no significant variation between the measurements of outer opisthosomal setae from adult males individual sample from different animals. The obtained data enhance the need for a good hygienic and management measures for control of the disease. Availability of genomic information of ITS2 gene may help in future discovering of Psoroptes natalensis sub-species and increase progress in developing of novel acaricidal drug targets.