A low pathogenic avian influenza virus of the H5N2 subtype was isolated for the first time from layer chickens in Japan in 2005. Surveillance in trading restriction zones and epidemiologically related farms revealed 41 seropositive farms, and 16 H5N2 viruses were isolated and characterized from nine of these farms. That these viruses were genetically and antigenically similar to each other suggested that these isolates were derived from a common origin. Complete genomic characterization of all eight gene segments showed that these H5N2 isolates in Japan had high homology to the H5N2 strains prevalent in Central America since 1994. The virus was reisolated from tracheal and cloacal swabs of experimentally inoculated chickens and efficiently transmitted to sentinel chickens in adjacent cages.