Background: Highly pathogenic avian influenza (HPAI) H5 viruses have exhibited substantial geographic spread throughout Asia, Europe, Africa, and recently North America since their first detection in 1996. Accumulation of mutations in the HA gene has resulted in several phylogenetic clades, while reassortment with other avian influenza viruses has led to the emergence of new virus subtypes (H5Nx), notably H5N2, H5N6, and H5N8. H5Nx viruses represent a threat to both the poultry industry and human health. As reassortment and accumulation of mammalian adaptations may increase virus fitness and virulence, continuous in vivo evaluation of H5Nx viruses is necessary in aiding pandemic preparedness efforts. Methods and materials: Ferrets were inoculated with HPAI H5N6 virus (A/Sichuan/26221/2014, A/Yunnan/14563/2015, or A/duck/Bangladesh/19D770/2017) and evaluated for clinical signs and symptoms of infection, replication throughout the respiratory tract, extra pulmonary virus spread, and virus transmission. A North American HPAI H5N2 virus (A/turkey/Minnesota/10915/2015) was used for comparison. Levels of virus released into the air by infected ferrets was evaluated using a cyclone bioaerosol sampler. Replication kinetics of all viruses in the human bronchial epithelial cell line, Calu-3, was evaluated. Results: Tested H5N6 viruses were more pathogenic in ferrets compared to the H5N2 virus. However, pathogenicity profiles of the H5N6 viruses varied by strain, from a mild infection with only sporadic virus dissemination beyond the respiratory tract, to a severe and fatal infection. In vitro evaluation of H5Nx virus replication in Calu-3 cells and the presence of mammalian adaptation markers in key genes supported these findings. Limited transmission between co-housed ferrets was observed with the H5N6 viruses but not the H5N2 virus, which is in agreement with generally low levels of aerosolized virus exhaled from virus-inoculated ferrets. Conclusion: HPAI H5 viruses bearing the N6 subtype neuraminidase and mammalian adaptation markers in the polymerase genes are capable of causing lethal infection in ferrets as well as transmitting to uninfected animals in a direct contact setting. The heterogeneity in mammalian virulence among H5N6 viruses underscores the necessity of in vivo evaluation of H5Nx viruses as they continue to evolve and pose a threat to human health.