Tests confirmed the H5 strain of the virus at a farm in Yamaguchi prefecture on the southwestern tip of Japan's main Honshu island after its owner reported late Monday that several chickens had died suddenly, the farm ministry said.
Officials began the slaughter on Tuesday and asked farms within a six mile radius not to transport their poultry outside of the area.
The latest confirmed case comes a day after the government ordered the slaughter of about 42,000 chickens at a poultry farm in Miyazaki prefecture in southern Kyushu.
"We will continue to take all necessary measures if any further outbreaks are confirmed," Chief Cabinet Secretary Yoshihide Suga, the government's top spokesman, said at a ministerial meeting concerning the government's outbreak response.
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Japan's agriculture ministry regularly warns farmers about the risk of infection, citing the continued spread of the disease in Asia, including in South Korea.
Some strains of avian influenza are fatal for chickens, and pose a health threat to humans, who can fall sick after handling infected poultry.
Another strain of bird flu, H7N9, has claimed more than 170 lives since emerging in 2013.