Egg drop syndrome (EDS) is caused by a hemagglutinating adenovirus which has become a major cause of lost egg production and sever economic losses in breeder and layer chicken flocks throughout the world. A PCR assay was optimized for detection of EDS virus in inoculated allantoic fluids (AFs) of duck and chicken emberyonated eggs. Two strains of EDS viruses were propagated in allantoic cavity of eggs. Then the virus DNA was extracted. PCR test was designed and carried out by specific primers. The 1900 bp band was detected in agar gel electrophoresis. Serial two fold dilutions of infected allantoic fluid were prepared. The HA test and PCR assay were carried out for each dilution and finally results were compared to each other. The PCR assay could detect some negative HA titers. This study clearly indicates the superiority of PCR assay over HA test for detection of EDS virus in AFs.