The aim of this study was to evaluate the hypothalamic-pituitary-adrenal (HPA) axis activity of Holstein-Friesian and crossbreed F1 heifers by analysis of the cortisol concentrations in hair samples. Cortisol, the primary hormone of the HPA axis, is the biological endpoint for the investigation of the HPA response. The study was conducted on 290 prepubertal heifers; 142 heifers were pure Holstein-Friesian and 148 were crossbreed F1 heifers obtained from the 3-way rotational system with Swedish Red and Montbéliarde breeds. Extraction was performed on the hair using methanol, and cortisol concentrations were determined by a radioimmunoassay method. Cortisol concentrations measured in regrown hair of crossbreed F1 heifers were significantly lower than those in hair of Holstein-Friesian heifers. This result helps us to better understand the differences in HPA activity and allostatic load between Holstein-Friesian and crossbreed F1 heifers and allows us to better assess the adaptability of these animals to the environment and the importance of crossbreed traits for profitability in dairy farming.