Since 2013, highly pathogenic H5N6 avian influenza viruses (AIVs) have emerged in poultry and caused sporadic human infections in Asia. The recent discovery of three new avian H5N6 viruses – A/oriental magpie-robin/Guangdong/SW8/2014 (H5N6), A/common moorhen/Guangdong/GZ174/2014 (H5N6) and A/Pallas's sandgrouse/Guangdong/ZH283/2015 (H5N6) – isolated from apparently healthy wild birds in Southern China in 2014–2015 raises great concern for the spread of these highly pathogenic AIVs (HPAIVs) and their potential threat to human and animal health. In our study, we conducted animal experiments and tested their pathogenicity in ducks, chickens and mice. Ducks can carry and shed the H5N6 HPAIVs, but show no ill effects. On the other hand, these H5N6 HPAIVs can efficiently infect, transmit and cause death in chickens. Due to the overlap of habitats, domestic ducks play an important role in circulating AIVs between poultry and wild birds. Our results raise the possibility that wild birds disseminate these H5N6 HPAIVs to poultry along their flyways and thus pose a great threat to the poultry industry. These viruses are also highly pathogenic to mice, suggesting they pose a potential threat to mammals and, thus, public health. One virus isolated in 2015 replicates much more efficiently and is more lethal in these animals than the two other viruses isolated in 2014. It seems that the H5N6 viruses tend to be more lethal as time passes. Therefore, it is necessary to vigilantly monitor H5N6 HPAIVs in wild birds and poultry.