Honeybee defensive behaviour is an important trait for selection of honeybees for breeding programs. We evaluated the variation in honeybee defensive behaviour with environmental factors and hive conditions. Factors such as the difference in the agro-ecological zones, colony strength, mean elevation, type of bee hive used, and the vegetation cover were considered. The number of honeybees attacking the researchers’ protective gear within one minute of disturbance was recorded per colony and analyzed. Apis m. adansonii was found to be the most defensive. Variations in the agro-ecological zones, colony strength, and mean elevation were found to significantly influence the defensive behaviour of the honeybees. Honeybee colonies in the Mid North AEZ were the most defensive. The type of bee hive and vegetation cover did not have any influence on the defensive behaviour. From this study, we suggest that selection of honeybees that are less defensive for breeding programs should consider A. m. scutellata and honeybee colonies from West Nile and Southern Highland AEZs at higher elevations.