Social insects provide a useful model for studying the evolutionary balance between cooperation and conflict linked to genetic structure. We investigated the outcome of this conflict in the bumblebee, Bombus terrestris, whose annual colony life cycle is characterized by overt competition over male production. We established artificial colonies composed of a queen and unrelated workers by daily exchange of callow workers between colony pairs of distinct genetic make-up. Using microsatellite analysis, this procedure allowed an exact calculation of the proportion of worker-derived males. The development and social behavior of these artificial colonies were similar to those of normal colonies. Despite a high worker reproduction attempt (63.8% of workers had developed ovaries and 38.4% were egg-layers), we found that on average 95% of the males produced during the competition phase (CPh) were queen-derived. However, in four colonies, queen death resulted in a considerable amount of worker-derived male production. The different putative ultimate causes of this efficient control by the queen are discussed, and we suggest a possible scenario of an evolutionary arms race that may occur between these two female castes.