This study determined cortisol concentrations in hair that had grown for one month and in hair from a previously unshorn area and examined the effects of calendar month, pregnancy and illness on hair cortisol concentrations in dairy cows. The study was conducted over a one-year period using 27 cows. Electric clippers were used to collect two hair samples per cow each month. The first sample (A sample) consisted of hair that had grown for one month in a pre-clipped area and the second sample (B sample) comprised all hair from a previously unshorn area. Liquid chromatography tandem mass-spectrometry was used for cortisol measurement. The overall mean concentrations for A and B samples did not differ. Cortisol concentrations of A samples were significantly higher in the winter (0.86±0.37pg/mg) than in the fall (0.67±0.33pg/mg). The hair cortisol concentration in A samples increased during pregnancy and the maximum concentration of 1.40±1.08pg/mg hair in the month of calving was significantly higher than the concentration measured in the first month (0.66±0.32pg/mg). The findings show that the effect of short-term stressors such as parturition on hair cortisol concentrations are more easily detected in hair that has grown for one month than in hair from a previously unshorn area.