Sheep pox (SP) is a serious skin disease of sheep. Accurate and rapid diagnosis of SP is very important to control the rapid spread of disease in Egypt. In this study seven skin scabs from infected sheep at different localities in Menofeia province, Egypt were used for isolation and identification of SP virus. A total of 200 serum samples were collected from non-vaccinated and vaccinated sheep to detect the neutralizing antibodies for SP virus by serum neutralization test (SNT). Isolation of SP virus was done by inoculation on chorioallantoic membrane (CAM) of specific pathogen free embryonated chicken eggs (SPF-ECEs). Identification of SP virus isolate was confirmed by immunoflourescence (IF) and polymerase chain reaction (PCR) targeting SP virus PRO30 gene. In conclusion, SP virus is still circulated in Egypt and a test for differentiation between infected and vaccinated sheep with SP virus is required.