Cortisol is commonly used as an indicator of physiological stress. Traditional samples (blood, urine, and saliva) used to measure cortisol secretion have proven to adequately assess short-term/acute response to stress, while hair cortisol concentration (HCC) is increasingly being used to assess long-term stress in mammals. The aim of this study was to quantify HCC in whole hair and hair segments to investigate potential effects of body location and surgical procedure (castration) on HCC in horses. A preliminary study was performed to investigate potential differences in HCC between collection site (mane and tail). Then, effects of castration on HCC were assessed. Preliminary study: mane and tail hair from healthy broodmares (n = 10) was collected, segmented, and analyzed. Castration study: yearling male (n = 14) and female horses (n = 14) were used. Mane and tail hair were collected at the beginning of the study (before castration) and 3 months after. For both studies, cortisol was extracted into methanol from ground hair and quantified using ELISA. Hair cortisol concentration in mane samples from broodmares was greater than in tail samples. In all groups, segments further away from the hair root contained lower HCC. The decrease in HCCs between 1- and 2-month postcastration was significantly lower in castrated males than intact females. Cortisol accumulation in the month after castration could explain the significantly smaller decrease in HCC in males than females during the same period. Results suggest that HCC analysis in horses can be used to address basal and elevated hypothalamic–pituitary–adrenal activity in horses. Nevertheless, other factors (segment location and collection site) may also have an effect on measured HCC.