To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP (I)109(T)). By analyzing viruses constructed by the reverse-genetic method, we established that the NP (I)109(T) substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP (I)109(T) substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP (I)109(T) substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP (I)109(T) mutation would be induced frequently during replication of HPAIVs in brains, but not in lungs. These results demonstrate that the amino acid at position 109 in NP enhances viral RNA synthesis and the pathogenicity of highly pathogenic avian influenza viruses in chickens and that the NP mutation emerges quickly during replication of the viruses in chicken brains.