Simple SummaryHoney bee aggression constitutes an important defensive mechanism for honeybee colonies and humans. However, due to the complexity associated with this behavior, beekeepers are still faced with the challenges of selecting defensive and less-defensive bred lines. This could be achieved through selective breeding in closed mating stations. We endeavored to evaluate defensiveness and orientation in five bred lines of honeybee colonies using a combination of chemical (alarm pheromone and isopentyl acetate mixed with paraffin oil) and physical (dark leather suede, colony marbling, and suede jiggling) stimuli to identify defensive and less-defensive bred lines. We found that chemical assays associated with physical stimuli recruited bees to the suedes, but the time of recruitment was faster when stimulated by alarm pheromone compared to isopentyl acetate mixed with paraffin oil. Defensiveness varied among bred lines, and orientation was highest in defensive bred lines compared to less-defensive bred lines. Beekeepers are therefore advised to evaluate orientation defensiveness at the colony level and among bred lines when selecting breeding colonies for defensive purposes.AbstractHoney bees (Apis melliferaL.) express complex behavioral patterns (aggressiveness) in defensive mechanisms for their survival. Their phenotypic expression of defensive behavior is influenced by internal and external stimuli. Knowledge of this behavior has recently become increasingly important, though beekeepers are still faced with the challenges of selecting defensive and less-defensive bred lines. Field evaluation of defensive behavior among bred lines of honey bees is required to overcome the challenges. Chemical cues (alarm pheromone and isopentyl acetate mixed with paraffin oil) and physical and visual stimuli (dark leather suede, colony marbling, and suede jiggling) were used to evaluate defensiveness and orientation among five bred lines of honeybee colonies. Our results showed that both chemical assays recruited bees, but the time of recruitment was significantly faster for alarm pheromone. Honeybees’ response to both assays culminated in stings that differed among bred lines for alarm pheromone and paraffin when colonies were marbled. Honeybee orientation defensiveness varied among bred lines and was higher in more defensive bred lines compared to less-defensive bred lines. Our findings suggest that it is crucial to repeatedly evaluate orientation defensiveness at the colony level and among bred lines when selecting breeding colonies.