Most highly pathogenic avian influenza A viruses only cause mild clinical signs in ducks serving as an important natural reservoir of influenza A viruses. However, we isolated two H5N1 viruses that are genetically similar but differ greatly in the virulence in ducks. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is low pathogenic. To determine the genetic basis for the high virulence of CK10 in ducks, we generated a series of single-gene reassortants between CK10 and GS10 and tested their virulence in ducks. Expression of CK10 PA or HA gene in the GS10 context resulted in increased virulence and virus replication. Conversely, inclusion of GS10 PA or HA gene into the CK10 background attenuated the virulence and virus replication. Moreover, the PA gene has a greater contribution. We further determined that residues 101G and 237E in the PA gene contribute to the high virulence of CK10. Mutations at these two positions produced changes in virulence, virus replication and polymerase activity of CK10 or GS10. Position 237 plays a greater role in determining these phenotypes. Moreover, K237E mutation in GS10 PA gene increased the PA nuclear accumulation. Mutant GS10 viruses carrying CK10 HA gene or PA101G or PA237E mutation induced an enhanced innate immune response. Sustained innate response was detected in the brain rather than in the lung and spleen. Our results suggest that the PA and HA genes-mediated high virus replication and intense innate immune response in the brain contribute to the high virulence of H5N1 virus in ducks.