This study presents a local isolate of fowl pox virus (FPV) isolated and propagated from backyard naturally infected laying hens in El-Sharkia Governorate, Egypt, during the period of January to November 2017. Isolation and propagation were carried out from collected skin lesions on the chorioallantoic membrane (CAM) and chicken embryo fibroblast (CEF) with obtained 4^th passage virus titers 4.0 Log₁₀ EID₅₀/ml at the in CAM and 3.5 Log₁₀ TCID₅₀/ml in CEF respectively. They showed characteristic pock lesions of FPV and cytopathic effect (CPE) of FPV at the 3^rd passage on CAM and CEF, respectively. Virus neutralization test (VNT) results confirmed that the obtained isolate is FPV. Molecular characterization of (Sharkia2017/VSVRI) was performed with Polymerase chain reaction (PCR) to amplify 578 bp of P4b (fpv167) gene and 1150 bp of fpv140 gene. Sequence and phylogenetic analysis of both genes confirmed the relatedness of (Sharkia2017/VSVRI) isolate to sub-clade A1 of fowl pox viruses with 99.7-100% identity to fowl pox virus sequences published in GenBank.This study reports the antigenic and genomic characterization of the locally isolated FPV (Sharkia2017/VSVRI) using VNT and PCR confirmed by sequence analysis to help in the production of FPV tissue culture vaccine from the obtained local FPV after confirming its immune response as a candidate vaccine.