H4 and H9 avian influenza viruses (AIVs) are two of the most prevalent influenza viruses worldwide. The co-existence of H4 and H9 viruses in multiple avian species provides an opportunity for the generation of novel reassortants and for viral evolution. The diversity of the biological characteristics of the reassortants enhances the potential threat to the poultry industry and to public health. To evaluate the reassortment of these viruses and the potential public risk of the reassortants, we co-infected chickens with H4N6 and H9N2 viruses derived from poultry and tested the replication and virulence of the reassortant viruses in mice. A high frequency of reassortment was detected in chickens after co-infection with these two viruses and nine reassortants of six genotypes were purified from the chicken samples. Two H9N2 reassortants containing the PA of the parent H4N6 virus showed higher virulence than the parent H9N2 virus, revealing the significant role of the H4N6 wt virus PA gene in viral reassortment. Analysis of the polymerase activity of the ribonucleoprotein (RNP) complex in vitro suggested that the PA of H4N6 wt origin enhanced polymerase activity. Our results indicate that co-infection of an avian individual with the H4N6 and H9N2 viruses leads to a high frequency of reassortment and generates some reassortants that have higher virulence than the wild-type viruses in mammals. These results highlight the potential public risk of the avian influenza reassortants and the importance of surveillance of the co-existence of the H4N6 and H9N2 viruses in avian species and other animals.