A/H5N1 influenza viruses pose a threat to human and animal health. A fully avian A/H5N1 influenza virus was previously shown to acquire airborne transmissibility between ferrets upon accumulation of five or six substitutions that affected three traits: polymerase activity, hemagglutinin stability and receptor binding. Here, the impact of these traits on A/H5N1 virus replication, tissue tropism, pathogenesis and transmission was investigated in chickens. The virus containing all substitutions associated with transmission in mammals was highly attenuated in chickens. However, single substitutions that affect polymerase activity, hemagglutinin stability and receptor binding generally had a small or negligible impact on virus replication, morbidity and mortality. A virus carrying two substitutions in the receptor-binding site was attenuated, although its tissue tropism in chickens was not affected. This data indicate that an A/H5N1 virus that is airborne-transmissible between mammals is unlikely to emerge in chickens, although individual mammalian adaptive substitutions have limited impact on viral fitness in chickens.