To determine the pathogenicity of the viruses in chickens, we intranasally inoculated 6-week-old SPF white leghorn chickens with 105 EID50 of either H5N6 virus (i.e., SW8 or ZH283). All inoculated chickens exhibited clinical signs of illness, including severe depression, cloudy eye, and intermittent head-shaking, and died within 5 DPI, with a mean death time (MDT) of 3.3 to 4.0 d (Figure 1A). SW8 and ZH283 replicated systemically in chickens and at 3 DPI was detectable in all tested organs, including the respiratory tract (i.e., lung and trachea), kidney, lymphoid tissues (i.e., spleen, cecal tonsils, and bursa of Fabricius), pancreas, liver, brain, intestinal tract (i.e., duodenum, ileum, descending colon, and jejunum), and heart. SW8 and ZH283 replicated efficiently in the lower respiratory tract; high viral titers were detected in the lung, with mean titers of 6.33 log10EID50 and 8.58 log10EID50, respectively (Figure 1B). The two novel viruses also replicated in the brain, spleen, and bursa of Fabricius, with mean titers of 4.83–7.17 log10EID50, 5.83–7.33 log10EID50, and 6.08–7.58 log10EID50, respectively (Figure 1B). Overall, the two novel H5N6 influenza viruses of wild bird origin showed high pathogenicity in chickens and could replicate systematically in them.