H5N6 avian influenza virus (AIV) has posed a threat to poultry and human health. Here, we isolated a new clade 2.3.2.1 H5N6 virus, not like most reported H5N6 AIVs of clade 2.3.4.4. Our analysis revealed a complex pattern of its evolution in which the virus was derived as a result of genetic reassortment among clade 2.3.2.1 and clade 2.3.4.4 H5 and H6N6 AIVs. Moreover, the results suggested that H5N6 clade 2.3.2.1 AIVs had multiple reassortment pattern. It is necessary that to strengthen continuing surveillance to prevent infection in human and poultry.