FowlPox virus (FPV) was detected in eight chickens suffering from a diphtheritic lesion on the oropharynx and trachea with nodular skin lesions around the unfeathered parts in two Egyptian governorates (El-Sharkia and Ismailia governorates) during summer 2023. A variety of serological and molecular methods were performed for identification and characterization of the virus. on specific-pathogen free (SPF) embryonated chicken eggs via chorio-allantoic membranes (CAM), the distinguishing focal pock lesions were detected on CAM. Concerning electron microscopy, FPV appeared as enveloped quadrangular brick shaped Avipoxvirions. The neutralizing antibodies level against FPV were detected in all eight samples. Serum neutralization test showed a neutralization index of ≥ 1.6 in all serum samples, meanwhile ELISA test displayed an S/P ratio of ≥ 1.5 in the affected chickens. Notably, two positive FPV samples were sequenced then submitted to the GenBank (Sharquia-1 and Ismilia-2 with accession numbers; OR920788-OR920789). The phylogenetic tree construction based on the fpv167- (P4b) gene of FPV revealed high nucleotide identity with Elsharqyia_FWPV1, Elsharqyia_FWPV2 and Fowlpox-AN5, FWPVN, FWPVD (Egyptian isolates) with nucleotide identity percentage 100%, 99%, 100%, 99%,99%; respectively. Likewise, FPV isolates were of low homology with VSVRI-Vac (vaccinal strain) with 88% similarity. In context, the local recent our strains can be applied in vaccine production for appropriate vaccination programs in Egypt.