Two complete duck hepatitis virus type 1 (DHV-1) genomes, strain SY5 and its chicken embryos passage descendent vaccine strain ZJ-A, were compared and analyzed in order to identify possible sites of attenuation. Of the 205 nucleotide changes, 22 resulted in sense mutations, 174 produced nonsense mutations. Besides, there are 7 consistent nucleotides substitutions in 5'UTR and 2 in 3'UTR. Three of these 22 sense mutations resided in VP0, 6 exists in VP1, one exists in VP3, 3 exists in 2A2, 3 exists in 2C, one was detected in 3B and 5 was in 3D. These results suggested that VP0, VP1, 3D, and 5'/3'UTR may contribute to the attenuation of DHV-1 in chicken/duck/embryos. The results provide a genetic basis for future manipulation of a DHV-1 infectious clone.