This study aimed to determine hair cortisol concentrations (HCC) in various sampling sites on the body of lactating Holstein cows and heifers. Forty-seven multiparous lactating Holstein cows (DIM = 110 ± 47, BW = 623 ± 75 kg) and 23 Holstein heifers (10–12 mo of age, BW = 258 ± 20 kg) were used in this experiment. To obtain serum, blood was collected by jugular venipuncture in vacutainer tubes at 12:00 h at the d 0, after one month and after 2 mo. Hair sampling sites included forehead, withers and rump sites. Statistical analysis was carried out using the ANOVA procedure of SAS. The intra-assay and inter-assay coefficients of variations were 3.15 and 10.05, respectively. Serum cortisol showed high fluctuations between sampling times (d 0, 1 mo and 2 mo) whereas first sampling values were the highest in both lactating cows and heifers (P < 0.05). No differences were found in HCC among the various sampling sites in both lactating cows and heifers (P > 0.05). It is concluded that the distribution of cortisol into the hair shaft does not depend on hair sampling sites, and so hair sampling for cortisol analysis can be collected from any of the 3 regions based on the ease of collections and the location’s facilities.