We isolated a new flavivirus from Aedes albopictus mosquito and a related species in Japan. The virus, designated Aedes flavivirus (AEFV), only replicated in a mosquito cell line and produced a mild cytopathic effect. The AEFV genome was positive-sense, single-stranded RNA, 11,064 nucleotides in length and contained a single open reading frame encoding a polyprotein of 3341 amino acids with 5' and 3' untranslated regions (UTRs) of 96 and 945 nucleotides, respectively. Genetic and phylogenetic analyses classified AEFV with the insect flavivirus, but distinct from Cell fusing agent (CFA), Kamiti river virus and Culex flavivirus. Interestingly, a partial sequence of AEFV showed significant similarity to that of Cell silent agent (CSA), the insect flavivirus-related nucleotide sequence integrated in the genome of A. albopictus. These results suggest that AEFV is a new member of the insect flaviviruses, which are intimately associated with Aedes mosquitoes and may share a common origin with CSA.