Simple SummaryHair tissue records a longer activity profile of an animal’s hypothalamic–pituitary–adrenal axis, and resulting production of cortisol, compared to other matrices (e.g., blood serum, saliva, or feces). Cortisol is a key hormone involved in mobilizing stored energy reserves to assist in meeting physiological demands, but production can also be triggered by psychological stimuli or perceived demands. For horses (Equus ferus caballus), most studies investigating hair cortisol concentrations (HCC) have used domesticated individuals where nutritional requirements are fully met, social structures may not be as dynamic, or reproductive behaviors and physiological demands are inhibited. Here, we investigated HCC from 282 samples of hair collected from a population of feral horses that exist under fully natural conditions, including a self-determined social structure, at Sable Island, Nova Scotia, Canada. We found that HCC was predominantly associated with sex, age, body condition, and year effects but, also, for females, the presence or absence of a foal. Female HCC was further influenced by social factors, including harem size and the number of adult males not associated with a band (i.e., bachelors) that coexisted in their home range. In addition, we evaluated biological (e.g., hair color) and procedural factors (sample mass and hair texture) against observed HCC.AbstractCortisol is a glucocorticoid hormone produced during activation of the hypothalamic–pituitary–adrenal axis (HPA) in response to psychological or physiological demands. High amounts of circulating cortisol can be found in individuals experiencing energetically demanding physiological events, such as pregnancy, lactation, injury, or starvation, but, also, in individuals who may have less obvious HPA activation from social situations. The feral horse population on Sable Island (Nova Scotia, Canada) provides an opportunity to look at hair cortisol concentration (HCC) as a proxy for circulating cortisol concentration to better understand physiological correlates. The horse’s complex social structure also allows us to look at how the population and group structure may influence HPA activation. Hair samples (n= 282) were analyzed from 113 females and 135 males. Females with dependent offspring (foals) had higher HCC than those females without dependent offspring (p= 0.005). Horses in poor body condition were also more likely to have higher HCC (females:p< 0.001, males:p= 0.028); females had greater variation in the body condition index (BCI), which also correlated with foal production. In general, the top-ranked models describing female cortisol levels included age, BCI, presence of a foal, as well as social measures such as harem size and the number of bachelors in the vicinity. The top model describing male cortisol levels included age, BCI, and year of collection only, and the number of bachelors in the home range appeared in subsequent, though still high-ranked, models. Among the variables not of direct interest, we found some significant results relating to hair color and hair texture. Differences in HCC patterns between feral and domestically kept horses (e.g., age and sex) are likely linked to periods of resource limitations, particularly for individuals experiencing energetically demanding processes such as reproduction, illness/parasitism, or related to experiencing the full range of social and reproductive behaviors.