Abstract: A complete open reading frame (ORF) of UL35 gene encoding the protein VP26 of duck plauge virus (DPV) was obtained according to the DNA sequencing information of a recombinant plasmid of the DPV CHv strain gene library constructed in our laboratory combining together with the analysis of ORF Finder and BLAST tool of NCBI. Then the DPV VP26 gene was cloned into pMD18T，and was strongly confirmed by PCR amplification, restriction digestion and oligonucleotide probe hybridization. Molecular characterization analysis indicated that the DPV VP26 gene was composed of 354 nucleotides,and encoding a polypeptide of 117 amino acid residues, which had a Herpes_UL35 conserved domain related to small nucleocapsid protein family and highly conserved among the Herpes_UL35 proteins. Moreover, the nucleic acid and amino acid sequence of DPV VP26 had higher homology with its homologous protein of Alphaherpesvirus than others. Phylogenetic tree analysis showed that on taxonomic status the DPV could be grouped in Alphaherpesvirus subfamily, and might be a new genus of Alphaherpesvirus. Subcellular location analysis demonstrated that the VP26 mainly located in nucleus. Besides, condon preference analysis demonstrated that the alternative codons for the same amino acid in VP26 had distinctly different frequency and the DPV VP26 had 18, 19 and 25 codons’ frequency evidently disagreeing with E. coli, yeast and human, respectively. These results provide a molecular biology evidence for the further study on the function and mechanism of DPV VP26.