Duck hepatitis strains 90D and 04G were determined to be antigenically unrelated to type 1 duck hepatitis virus (DHV-1) by in vitro cross-neutralization assay. The genome sequences of 90D and 04G revealed that both strains of the new serotype DHV (N-DHV) possessed a typical picornavirus genome organization apart from the unique possession of three in-tandem 2A genes present in DHV-1. The 2A1, 2A2, and 2A3 proteins represented an aphthovirus-like 2A protein, AIG1-like protein, and human parechovirus-like 2A protein, respectively. The N-DHV genome displayed unique features, compared to the DHV-1 genome. The 366 nt 3'UTR of N-DHV, the largest determined thus far among picornaviruses, was 52 nt longer than DHV-1. The pairwise percent identity of the nucleic acid and amino acid sequences at 1D region of N-DHV and DHV-1 were only 69.1-69.7 and 70.1-70.5%, respectively. Finally, phylogenetic and evolutionary analysis of N-DHV revealed that the N-DHV and DHV-1 belong to two different clusters of a novel genus in the Picornaviridae family.