Technical Abstract: Highly pathogenic H5N1 avian influenza viruses produce severe disease and mortality in chickens. Identification of viral genes important for cell tropism and replication efficiency helps identify virulence factors. To determine which viral gene or genes contribute to the virulence of H5N1 avian influenza viruses in chickens, we used reverse genetics to generate single-gene recombinant viruses and examined their pathogenicity in chickens. Intranasal inoculation of two week-old chickens with the recombinant avian influenza virus rEgret/HK/02 resulted in 100% mortality and high viral titers in tissues. Inoculation of chickens with rCk/Indonesia/03 produced only 50% mortality with significantly less viral replication in tissues. Reassortants combining genes of the mentioned viruses demonstrated that exchanging the hemagglutinin and PB2 genes considerably affected pathogenicity; this was reflected in differences in mortality, viral replication, and spread in tissues. The NS gene also had an effect on viral replication and spread in tissues; however, no effect on mortality was observed. Differences in mRNA cytokine levels in lung and spleen were observed among the different groups, indicating a role of the innate immune response on the outcome of infection.