We used variation in the mitochondrial DNA (mtDNA) control region (D-loop) to examine the genetic structure of the sika deer (Cervus nippon) population on the Boso Peninsula, central Japan. A total of four haplotypes was found. In order to examine whether or not artificial barriers such as roads, dams, and golf courses affect the spatial heterogeneity of mtDNA haplotypes, we implemented two exclusive spatial analyses (SAMOVA and network analysis based on Monmonier’s algorithm) for searching genetic discontinuities between artificial barriers. Prior to the analyses, the whole distribution area was divided into meaningful eight blocks. Analysis of molecular variance (AMOVA) detected significant spatial heterogeneity in the constitution of the haplotypes among the blocks. The subsequent spatial analyses detected some significant spatial discontinuities on borders of the blocks. In particular, the largest discontinuity was observed in the area including motorway Line 81, but the traffic density of Line 81 is generally not very heavy compared to other major roads. These findings suggest that roads could be one of major barriers to hamper migration of sika deer to some extent, but other potential factors such as the location of food resources and/or the history of bottleneck event are also likely to more or less contribute to configure the present patterns of haplotype distribution.