Roe deer (Capreolus capreolus), as a representative of the Cervidae family, are particularly sensitive to negative environmental stimuli due to their need to maintain increased vigilance during feeding. Intensive hunting seasons are undoubtedly also a stressful factor for this species. The aim of this study was to analyze the cortisol levels in the hair of male roe deer collected at the beginning and the end of the hunting season in eastern Poland. In total, hair samples from 26 individuals collected in May and 31 individuals collected in September were analyzed. The concentration of cortisol in the hair was determined using the EIA kit. It was shown that the concentration of cortisol was significantly higher in samples collected in September compared to those collected in May (Pr. > |t| = 0.0017). Moreover, the age of animals and carcass mass did not significantly affect the concentration of the tested hormone. In summary, the concentration of cortisol in the fur of male roe deer depended on the season and was not influenced by the carcass mass or age of the animals studied.