Avian influenza A viruses of H5 subtype can cause highly pathogenic disease outbreaks in poultry. In March 2014, a new reassortant H5N6 subtype HPAI virus emerged in ducks and chickens in Lao People’s Democratic Republic. We have assessed the pathogenicity, pathobiology and immunological responses associated with this virus in chickens. Infection caused moderate to severe disease in 6 of 6 chickens within 48 hours following mucosal inoculation. High virus titers were observed in blood and other tissues taken at euthanasia. Viral antigen was demonstrated in endothelium, neural parenchyma, myocardium, lymphoid tissues and other cell types. Pro-inflammatory cytokines were greatly elevated compared to non-infected birds. Our study demonstrates that this new reassortant is highly pathogenic, causing disease in chickens similar to that of Asian H5N1. Its recent detection in Lao PDR, China and Viet Nam indicates that it may be an emerging threat to poultry and human populations.