The objective of this study was to evaluate the effects of chronical exposure of honeybee drones to environmental (5 ppb) and non-environmental concentration (200 ppb) of imidacloprid (IMD) on sperm concentration, motility, viability, and mitochondrial membrane potential measured in semen obtained from 180 drones originating from 18 colonies. The results demonstrate that IMD exposure did not affect sperm concentration; however, there were significant differences in concentration within colonies. IMD exposure was associated with reductions in sperm motility, which also varied within colonies. Statistically significant interactions between IMD exposure and colony were found for active mitochondria and sperm viability. Our results strongly suggest that neonicotinoids can negatively affect honeybee drone sperm quality. It is important to emphasize that IMD actions can be strongly modulated according to the colony.