To assess the genetic diversity of the red fox (Vulpes vulpes) population on Hokkaido Island, northern Japan, we examined genotypes of exon 2 of the major histocompatibility complex (MHC) class II DRB1 gene for 232 individuals and identified 17 novel alleles. The subpopulation in the Southern area was genetically differentiated from those in all other areas on Hokkaido, suggesting isolation in southern Hokkaido. In addition, the Southern subpopulation is lower in genetic diversity than the other subpopulations, possibly resulting from purifying selection and/or a recent bottleneck. The non-synonymous substitutions exceeded the synonymous substitutions for codons encoding antigen-binding sites (ABSs) in exon 2, indicating that the red fox DRB1 alleles have evolved under positive selection. In a Bayesian phylogenetic tree, although most of the DRB1 alleles from the Hokkaido red fox were contained within a red fox-like canid (Vulpes) clade, some belonged to another canid clade. This means trans-species polymorphism maintained by balancing selection. Our results showed the local variability and the presence of selection on the MHC gene in this population, which contributes to the understanding of the historical background and the molecular evolution.