During the 2020-2021 winter, Eurasian countries experienced large outbreaks caused by the clade 2.3.4.4b H5N8 subtype high pathogenicity avian influenza viruses (HPAIVs) in the wild bird populations. At least seven gene constellations have been found in the causal HPAIVs. When and where the various HPAIVs emerged remains unclear. Here, we successfully cloned H5N8 HPAIVs with multiple gene constellations from a tracheal swab of a dead mallard found at its wintering site in Japan in January 2021. According to their phylogeny, the bird was most likely co-infected with the E2 and E3 genotype clade 2.3.4.4b HPAIVs. The result indicates that feral waterbirds can be infected with multiple HPAIVs, and shed an HPAIV with novel gene constellation in Southern wintering sites.