This study was performed to investigate the effect of mixed rearing of male and female chickens on the stress response in Korean native chickens. To identify the degree of the stress response, heterophil-lymphocyte ratio (H/L ratio), heat shock protein genes (HSPs) expression, and intracellular nuclear DNA damage rate were analyzed before and after the mixed rearing of male and female chickens. The results showed that the H/L ratio of chickens after mixing males and females was more than thrice as higher than before mixing (<i>P</i><0.001), but the differences between males and females were not significant. HSP-70, HSP90-α, and HSP90-β expression levels were 2.5 to 3.4 times higher after mixing male and female chickens, compared to before mixing (<i>P</i><0.01). In the mixed rearing of male and female chickens, the increase in HSPs expression in females was higher than in males. Comet indicators in intracellular DNA damage rate analysis showed a significant increase after mixing male and female chickens compared to before mixing (<i>P</i><0.001). However, there was no significant difference between males and females with respect to DNA damage rate. Taken together, these results suggest that male and female mixed rearing acts as a strong external stressor in both male and female chickens.