Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. Rodent species which are potential hosts for TBEV are widely distributed in various regions in Japan. In this study, we carried out large-scale epizootiological surveys in rodents from various areas of Japan. A total of 931 rodent and insectivore sera were collected from field surveys. Seropositive rodents against TBEV were found in Shimane Prefecture in Honshu, and in several areas of Hokkaido Prefecture. These results emphasize the need for further epizootiological and epidemiological research of TBEV and preventive measures for emerging tick-borne encephalitis in Japan.