The effects of a few decades of habitat fragmentation on the population genetic diversity of the large Japanese field mouse, Apodemus speciosus, in the forest region around Fukuyama University was assessed with approximate 300 bp nucleotide sequences of the mitochondrial D-loop region. Two independently isolated internal populations within the campus showed a smaller number of haplotypes and lower haplotype and nucleotide diversity indices compared to those inhabiting a wide external forest area. Based on the Hst index, significant genetic differentiation was observed between geographically closely residing external and internal populations but not between distantly residing external populations, suggesting the presence of strong physical barriers between the external and internal populations. Fine-scale changes in the landscape configuration recorded in aerial photographs of Fukuyama University revealed plausible physical barriers and a correlation between isolation time and genetic diversity. The results suggest that the population genetic properties of forest dwelling mammals are sensitive to a few decades of short-term forest fragmentation created by artificial construction. Exploitation by humans should be conducted in harmony with wildlife ecology, such as by maintaining corridors, to achieve a wide habitat area for gene flow.