Cortisol levels were measured in hair, blood and milk in two different cattle breeds, kept under different breeding conditions and with different genetic merit for milk production. Cows and heifers of Holstein and Busha breeds were selected for the study. Cortisol concentration was determined by immunoassays. Cortisol accumulation was determined in proximal (close to the skin) and distal (far from the skin) segments of the hair shaft. The influence of hair colour and washing prior to extraction and analysis was also examined in order to establish additional factors that may have an impact on hair cortisol concentrations. Concentrations of cortisol determined in the proximal and distal segments of the shaft were significantly higher in Holstein than Busha cows and heifers (P<0.05 and P<0.01, respectively). In Holstein cows, no significant difference was found between concentrations in black and white hair. In hair washed with isopropanol, cortisol concentration was significantly lower compared to unwashed hair (P<0.01). Thus, cortisol concentration in hair varies with the technique of hair processing (washing), but not with colour in Holstein cows. Blood serum cortisol concentrations in Holstein cows and heifers were significantly higher than in Busha cows and heifers, (P<0.01 and P<0.05, respectively). Milk cortisol in Holstein cows was significantly higher than in Busha cows (P<0.05). The higher cortisol concentrations in Holstein cows are assumed to be the result of intensive breeding and physiological adaptation to high milk production.