The European bumblebee, Bombus terrestris, is an invasive eusocial species whose distribution is expanding greatly beyond its native range because numerous colonies are imported to or locally produced in non-native countries for pollination of agricultural crops. Closely related species exist in Japan where the unrestricted import and use of B. terrestris has resulted in the establishment of wild colonies. Laboratory studies previously showed that B. terrestris and Japanese native species can copulate and produce fertilized eggs. Although these eggs do not hatch, the interspecific mating can cause a serious reproductive disturbance to native bumblebees. In this study, we determined the frequencies of interspecies mating between B. terrestris males and native bumblebee queens in the wild on the islands of Hokkaido and Honshu by analyzing the DNA sequences of spermatozoa stored in spermathecae of native queens. We found that 20.2% of B. hypocrita hypocrita queens and 30.2% of B. hypocrita sapporoensis queens had spermatozoa of B. terrestris males in their spermathecae. Given that a Bombus queen generally mates only once in her life, such high frequencies of interspecific mating with B. terrestris pose serious threats to the populations of native bumblebees in Japan.