Abstract: To accurately and rapidly differentiate between the virulent and the attenuated vaccine strains of duck enteritis viruses (DEVs), sequences of the virus UL2 genes were retrieved from the databank at GenBank for the study. A 528 bp continuous nucleotide deletion region was found on the genes of both strains. Using the primer design software, Oligo 7.0, specific primers targeting for the development and optimization of the PCR differentiation diagnosis methodology based on the UL2 characterization were selected. The optimal annealing temperature was determined to be 55℃ rendering amplified segments for the DEV virulent and the vaccine strains at 1 019 bp and 491 bp, respectively. The established protocol was sensitive with a low detection limit of 15.3 pg; and, specific without cross-amplification with common duck origin pathogens, such as, Muscovy duck parvovirus, duck circovirus, goose parvovirus, Escherichia coli, Rimerella anatipstifer and Pasteurella multocida. Consequently, it was considered applicable for future studies on the pathogenic mechanism of DEV.