To investigate the possible spread of West Nile virus (WNV) into Japan, we carried out entomological surveillance for flaviviruses at migratory bird stopover sites in Hokkaido, Japan, during 2003–2006. A total of 3,826 mosquitoes, identified as 15 species in five genera, were collected and 2,465 of these were grouped into 123 pools that were assayed for cytopathic effects on mosquito and mammalian cell cultures and for flavivirus RNA by reverse transcription-polymerase chain reaction using flavivirus universal primer sets for fragments of the NS3 and NS5 genes. Neither WNV nor other mosquito-vertebrate transmitted flaviviruses were detected in mosquitoes collected at any of the sites in Hokkaido, but five Culex flaviviruses and one novel Aedes galloisi flavivirus were identified from Culex pipiens L. s. l. and Aedes galloisi Yamada, respectively. Genetic and phylogenetic analyses based on the partial NS5 nucleotide sequences classified Aedes galloisi flavivirus with the insect flavivirus, but distant from Cell fusing agent, Kamiti river virus, and Culex flaviviruses, showing <74% sequence identities. Polymerase chain reaction-based bloodmeal analysis of 79 females showed that all of the Aedes and Ochlerotatus mosquitoes fed on mammals (deer and humans), whereas, Cx. pipiens s. 1. mosquitoes fed on both of avian (ducks and sparrows, 85.7%) and mammalian hosts (dog, 14.3%). We suggest that to date WNV has not become established in Japan.