Until 2002, H5N1 highly pathogenic avian influenza (HPAI) viruses caused only mild respiratory infections in ducks. Since then, new viruses have emerged that cause systemic disease and high mortality in ducks and other waterfowl. Studies on HPAI virus pathogenicity in ducks have been limited, and there is no clear explanation of why the pathogenicity of some H5N1 HPAI viruses has increased. The nonstructural protein 1 (NS1 protein) is known to suppress immune responses in influenza virus–infected hosts affecting virus pathogenesis. In order to determine if the NS1 protein contributes to increased virulence in ducks, single-gene reassortant viruses were generated. Exchanging the NS genes from A/Ck/HK/220/97 (a virus that produces mild disease in ducks) and A/Dk/VN/201/05 (a very virulent virus for ducks) in the rEgret/02 background (a recombinant virus derived from A/Egret/HK/757.2/02, a highly pathogenic virus in ducks) resulted in decreased mean death times compared to infection with the rEgret/02 virus in ducks, but the change was not statistically significant. Infection with the reassortant viruses affected the expression of immune-related genes in spleens and lungs when compared to controls, but when compared among them, the expression of the duck genes was similar. Furthermore, virus titers in spleen, lung, and brain as well as antigen distribution in various tissues were similar in ducks infected with the reassortant viruses. All together these data show that, under these experimental conditions, exchanging the NS gene had minimal effect on the virus pathogenicity, and it suggests that other viral genes, or combination of genes, are most likely contributing to the increased virulence of H5N1 HPAI viruses in ducks.