A seroepidemiological survey of Neospora caninum infection among dogs in Japan was conducted using species-specific enzyme-linked immunosorbent assay with recombinant surface antigen (Nc-SAG1t). Among 1,206 dogs examined, 126 dogs (10.4%) from 30 prefectures from Hokkaido to Okinawa were positive to N. caninum infections, which were more frequently detected in females than males. Siberian Huskies showed the highest positive rate compared with the other breeds. Dogs with pyometra and diabetes mellitus showed the higher positive rates than dogs with other diseases or without diseases.