Outbreaks of H5N1 subtype highly pathogenic avian influenza virus (HPAIV) were recorded in chickens, domesticated birds and wild birds throughout Japan from November 2010 to March 2011. Genetic analysis of the Japanese isolates indicated that all gene segments, except the PA gene, were closely related to Japanese wild bird isolates in 2008 and belonged to clade 2.3.2.1 classified by the WHO/OIE/FAO H5N1 Evolution Working Group. Direct ancestors of the PA gene segment of all Japanese viruses analyzed in this study can be found in wild bird strains of several subtypes other than H5N1 isolated between 2007 and 2009. The PA gene of these wild bird isolates share a common ancestor with H5N1 HPAIVs belonging to clades 2.5, 7 and 9, indicating that wild birds were involved in the emergence of the current reassortant 2.3.2.1 viruses. To determine how viruses were maintained in the wild bird population, two isolates derived from chickens (A/chicken/Shimane/1/2010, Ck10 and A/chicken/Miyazaki/S4/2011, CkS411) and one from a wild bird (A/mandarin duck/Miyazaki/22M-765/2011, MandarinD11) were compared in their ability to infect and be transmitted to chickens. There was a significant difference in the survival of chickens that were infected with 10(6)EID(50) of CkS411 compared to those with MandarinD11 and the transmission efficiency of CkS411 was greater than the other viruses. The increased titer of CkS411 excreted from infected chickens contributed to the improved transmission rates. It was considered that reduced virus excretion and transmission of MandarinD11 could have been due to adaptation of the virus in wild birds.