Powassan (POW) virus is responsible for central nervous system infection in humans in North America and the eastern parts of Russia. Recently, a new flavivirus, deer tick (DT) virus, related to POW virus was isolated in the United States, but neither its pathogenic potential in human nor the taxonomic relationship with POW virus has been elucidated. In this study, we obtained the near-full-length genomic sequence of the DT virus and complete sequences of 3 genomic regions of 15 strains of POW-related virus strains. The phylogeny revealed 2 lineages, one of which had the prototype POW virus and the other DT virus. Both lineages can cause central nervous system infection in humans. By use of the combination of molecular definition of virus species within the genus Flavivirus and serological distinction in a 2-way cross-neutralization test, the lineage of DT virus is classified as a distinct genotype of POW virus.