Avian poxvirus (APV) is a global disease that affects birds, resulting in both pathological and financial losses for the poultry industry. It belongs to genus Avipoxvirus under the family Poxviridae. Viral lesions typically occur in the featherless parts at the head and around the eye. Samples were collected from six commercial layer chicken populations in the Al-Sharkyia governorate, Egypt during the period from May to June 2021. The virus was successfully detected by PCR targeting P4b gene at 578 bp and gave positive results in 5 out of 6 samples. Fowl Poxvirus infection was characterized by histopathology and isolation on specific pathogen free-embryonated chicken eggs (SPF-ECEs) followed by serological identification of the viral isolates using AGPT. Phylogeny of a 300 bp of P4b gene showed a high homology (99.99%) of of our isolates with most of local reference strains and vaccinal FPV strain from Egypt and lower homology with other Avian poxvirus strains from other species and were grouped within subclade A1 within Clade A in the phylogenetic tree. Amino acid sequence analysis of Avipoxvirus-Egy-f1086-2-P4b (OQ385208) acquired mutations as E120K and N121D while Avipoxvirus-Egy-f1086-3-P4b (OQ385207) acquired mutations as H83P and S93N (Yehia et al., 2023). Therefore, more research is needed to determine the effectiveness of the current vaccine against FWPV.