Sheep pox virus (SPPV) is one of members of genus Capripoxviruses in the Poxviridae family. It is considered a threat affecting sheep and causes significant financial losses in agriculture economy all over the world. This study was designed for isolation and molecular detection for SPPV from skin nodules of clinically suspected sheep in Kaliobyia province. Direct detection of the virus was carried out also on milk samples (n=25) as good entrants for early detection of SPV before the onset of clinical signs. Molecular detection and phylogenetic analysis of SPPV were performed based on open reading frame (ORF) 103 gene. Suspected skin nodules were used for virus isolation on Chorioallantoic membrane (CAM) of specific pathogen free embryonated chicken eggs (SPF-ECEs) for three successive passages. Two virus isolates and two milk samples were identified from the tested samples by polymerase chain reaction (PCR) using primers targeting ORF 103 (with an amplicon size of 570bp). Furthermore, one milk samples PCR product was sequenced and phylogenetically analyzed based on ORF103 gene and revealed that our new isolates was identical to Sheeppox virus isolated from Minufiya governorate with 97.1% homology. Also, it showed quite identity with other reference sequences of SPPV strains retrieved from the GenBank database. This study may afford to fresh insights about the epidemiological aspect of the circulating SPV circulating in Egypt.