Sheep pox virus (SPV) is a member of the genus Capripoxvirus, of family Poxviridae, which affect sheep and goats causes significant economic losses. The present study was applied for isolation and identification of Sheep pox virus from clinically affected sheep during 2017 to 2019 in five Egyptian governorates, El Wadi Al-Gadid, Marsa -Matrouh, Giza, Kafr El-Sheikh and Menufya. Despite the obligatory use of sheep pox vaccine for vaccination of sheep and goats in Egypt, but some factors hinder the mass vaccination for all sheep and goats' population like the grazing nutritional behaviour, poor management, scare feeding and inadequate veterinary services. The virus was isolated from skin lesions and lymph nodes on chorio allantoic membrane of Embryonated Chicken Eggs (ECE), with typical pock lesions, followed by identification on lamb testis culture (LT) showing the characteristic cytopathic effect. Similarly, the convential PCR depending on P32 gene, and by multiplex PCR give the same results proving that the isolated virus is SPV.