The genetic diversity of bumblebees can be adversely affected by habitat degradation. An overabundance of deer has altered the composition and diversity of herbaceous plants in many places of the world, resulting in decreases of herbaceous flowers. Populations of Bombus diversus may be strongly affected by this degradation of habitat in the Ashiu primary beech forest in Kyoto, Japan. To estimate the effects of deer browsing on B. diversus populations, we analyzed and compared the genetic diversity of the extant population in Ashiu to museum specimens collected prior to heavy deer browsing in Ashiu (1980s) and the extant population in Hyonosen primary beech forest in Tottori, Japan, which has not been as severely degraded by deer. We successfully amplified DNA from similar to 20-year-old museum specimens and determined the genetic diversity of B. diversus in Ashiu populations from the 1980s. Results were analyzed for indications of a bottleneck as well as estimates of N (e), allelic richness, rare allelic richness, expected heterozygosity, and the effective number of alleles. Our findings did not reveal clear evidence of degradation in genetic diversity of the extant Ashiu population compared to the museum specimens or to the Hyonosen population. Thus, the Ashiu population of B. diversus appears to have maintained a level of genetic diversity during 20 years irrespective of habitat degradation and the levels have been similar to that of the Hyonosen population.