Using reverse genetics (rg), we generated two reassortant viruses carrying the NS1 gene of two closely related HPAIV and LPAIV H7N1 variants (designated rgH7N7 HP(HPNS1) and rgH7N7 HP(LPNS1), respectively) in the backbone of the HP H7N7 strain A/Chicken/Netherlands/621557/03 (rgH7N7 HP). Comparison of these reassortants allowed us to determine the effect of amino acid differences in the nuclear export and nucleolar localization sequences of NS1 on pathogenesis in chickens. Compared to rgH7N7 HP(LPNS1), a delay in weight gain and an increase in mortality were observed for rgH7N7 HP(HPNS1). Furthermore, an increase in viral load in brains, lungs, and cloacal swabs, as well as an increased induction of mRNA for type I interferons and pro-inflammatory cytokines in brains, were observed for rgH7N7 HP(HPNS1). Comparison of rgH7N7 HP(LPNS1) with the backbone strain rgH7N7 HP allowed us to examine differences in pathogenesis due to differences in NS1 alleles. rgH7N7 HP, which contained allele A of NS1 showed a higher in vitro replication rate and proved to be more virulent than the isogenic virus carrying allele B of NS1(rgH7N7 HP(LPNS1)). In addition, higher virus accumulation in the lungs and brains, and an increased induction of host gene responses, especially in the brains, were found for rgH7N7 HP compared to rgH7N7 HP(LPNS1). No large differences were observed in type I interferon expression in the lungs of chickens infected with any of the viruses, suggesting that differences in virulence due to differences in NS1 could be related to differences in the induction of pro-inflammatory cytokines in vital organs such as the brains.