The latest outbreak occurred in the Okayama prefecture at a farm in Takahashi, about 560 km west of Tokyo, where at least 30 chickens died last week.
Officials have begun sterilising the farm, said the official speaking on condition of anonymity, citing protocol.
Local officials plan to start culling all 12,000 birds at the farm as early as Tuesday.
Thousands of chickens were killed at poultry farms in southern Miyazaki following two outbreaks of H5N1 bird flu in separate towns earlier this month.
To date, Japan has confirmed only one human H5N1 infection, and no human deaths.
The H5N1 virus has spurred the slaughter of millions of birds across Asia since late 2003, and caused the deaths of at least 163 people worldwide, according to the World Health Organization.
H5N1 can be transmitted through contact with infected birds’ waste but on the whole remains hard for humans to catch.
Health experts, however, fear that it may mutate into a form that could spread easily among humans and cause a pandemic.