In the present study, 10 pooled samples including nodules and diphtheritic membranes were collected front vaccinated and non-vaccinated chicken flocks at Dakahlia and Damietta governorates, Egypt during the period from June 2016 to August 2016. Trials for isolation of suspected virus from the collected samples were carried out via choriallantoic membranes (CAMs) of 10 days old embryonated chicken eggs (ECEs), collected from hens free from Fowl pox virus (FPV). Three serial egg passages were carried out for each sample. The isolated virus from field and 3rd egg passaged samples was identified by agar gel precipitation test (AGPT), indirect fluorescent antibody test (IFAT) and histopathological examination. Hyperimmune serum was prepared against standard FPV vaccine in rabbit. The identified virus was confirmed by polymerase chain reaction (PCR). The results of virus isolation revealed that out of 10 pooled samples, 7 samples were positive results after 1st passage, 8 samples were positive results after 2nd passage and 10 samples were positive results after the 3'd passage. The percentage of the positive results for field samples identified by AGPT and IFAT were 90% (9 out of 10 samples) and 100% (all 10 samples) respectively, while after 3rd egg passage was 50% (5 out of 10 samples) by AGPT and 100%) (all samples) by IFAT. The results indicate that IFAT and AGPT could be used for virus identification., but IFAT is more sensitive. Histopathological examination revealed specific eosinophilic intracytoplasmic inclusion bodies of FPV. PCR for field samples and the 3rd egg passages gave positive results in all samples targeting P4b gene at 578 bp, so PCR technique is more accurate and sensitive.