The American mink (Neovison vison) as invasive alien species has spread along the Chikuma River in Nagano Prefecture, central Honshu, Japan, through naturalization of animals escaped from fur farms in the past. To clarify genetic structures of the naturalized populations, we genotyped ten microsatellite loci on 126 wild-caught animals from this region. Consequently, the genetic variations of American minks in Nagano were relatively low, and no correlations between genetic distances and geographical distances were observed. Based on the genotype data, animals examined were divided to four genetically different clusters. The results suggest that the genetic characters of the American mink populations around the Chikuma River were not formed after naturalization, but during or before breeding in fur farms. In addition, the present study indicated their migration between localities which may bring gene flow and promote random mating, resulting in genetic structure change in the future populations.