Since the 1980s, the sika deer (Cervus nippon Temminck, 1838) population of Hokkaido, Japan, has grown, resulting in range expansion. To assess the effects of this range expansion on the spatial genetic structure of the population, we compared subpopulation structures during 2 different periods (168 samples for 1991-1996, and 169 samples for 2008-2010), using mitochondrial DNA (mtDNA; D-loop) and microsatellites (9 loci). The number of gene-based subpopulations decreased across the 15-year period; specifically from four to three subpopulations based on mtDNA, and from two to one subpopulation based on microsatellite DNA. The fusion of the two northern subpopulations caused the change to the mtDNA-based structure, which might be explained by the dispersal of females from higher to lower density subpopulations. In comparison, the reason for the change in the microsatellite DNA-based structure was unclear, because no significant genetic differentiation was observed between the two study periods. A stable mtDNA-based structure was maintained in the north and central population separated by a west-to-east boundary, while a north-to-south boundary in eastern Hokkaido maintained stability in the eastern subpopulation versus all other subpopulations. The findings of this study demonstrate the importance of understanding gene flow within a structured population to implement effective management efforts; for instance, the culling of one subpopulation might not affect an adjacent subpopulation, because deer movement is limited between the subpopulations.