Ecological genetic analyses have recently been applied to the field of biological invasions to describe the genetic background of the invasive species. The raccoon (Procyon lotor) is a medium-sized mammal that is native to North and Central America and has been naturalized into several countries worldwide. In the Boso Peninsula, Japan, raccoons were estimated to have been introduced during the 1990s. We collected DNA samples from 139 raccoons in seven administrative districts and sequenced a part of the mitochondrial D-loop region. We identified two haplotypes of the mitochondrial DNA, which differed by three single nucleotide polymorphisms; therefore, the raccoon population in Boso was founded by at least two females. Although the two haplotypes were identified in all districts, genetic differentiation analysis showed that the northern population was genetically different from the southern population. This genetic gap might not be affected by the landscape, but instead by the expansion history of raccoons. One of the probable hypotheses suggests that the expansion of raccoons may have occurred twice in Boso, a second expansion occurring after raccoons had already spread across Boso. Although this is consistent with historical distribution records, further studies using nuclear microsatellite markers are necessary to validate this hypothesis.