Technical Abstract: The virulence determinant for highly pathogenic avian influenza viruses is considered multigenic, although the best characterized virulence factor is the hemagglutinin cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also altering host gene expression levels. Disruption of the parental virus’ functional polymerase complexes by exchanging, PB1 or PB2 genes, decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the up-regulation of interferon and inducible Nitric Oxide Synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single gene reassortment events are sufficient to increase virulence of AIVs in chickens.