Simple SummaryHormone concentrations derived from hair can inform us of many aspects of an individual’s life, such as their reproductive status or social position. Cortisol is often associated with physiological or even psychological stress, while testosterone is strongly associated with male development and reproductive success. This study investigated cortisol and testosterone concentrations of tail hair collected from feral horses living in a naturally established social structure. We compare values of hair derived cortisol (n= 153) and testosterone (n= 48) to an individual’s social position as either an Immature male (2–4 years old), physically mature but non-reproductive male (Bachelor), reproductive dominant male (Stallion), or subordinate harem-associated male (tag). Immature males had significantly lower hair cortisol concentrations than adults. Hair testosterone concentrations were significantly higher in Stallions than males in the other social positions when age is accounted for. Bachelors exhibited a positive correlation between the two hormone levels, while among Stallions the association was negative. These findings suggest that patterns in hair hormone concentrations reflect known physiological interactions between cortisol and testosterone in relation to age, dominance, and reproduction, that have been established in the literature.AbstractSteroid-hormone concentrations from non-invasively obtained biomarkers, like hair, can provide a representation of circulating hormones diffused over relatively long time periods (e.g., weeks or months). The hormone cortisol is often associated with physiological or even psychological stress, while testosterone is strongly associated with male development and reproductive success. Increasingly, studies are using hormone levels derived from hair to make inferences among both domestic animals and wildlife. For horses, all previous hair hormone analysis has been done on companion or working animals. We evaluated the levels of hair cortisol (n= 153) and testosterone (n= 48) from 136 feral horses living on Sable Island, Canada that have been part of a long-term individual-based study since 2008. This population has been undisturbed and unmanaged for over 50 years, and exhibits the natural social organization for horses, harem defense polygyny. Hair samples were collected in mid to late summer and the segment analyzed corresponds with hair grown during, and following, the peak of the reproductive season. Social position was determined based on the male’s role as either a dominant breeding Stallion (Stallion), a non-breeding subordinate male (tag), adult Bachelor (5 years old or older), or Immature male (2–4 years of age). While there was no difference in hair-cortisol concentration among any class of adult males (i.e., Stallion, tag, or Bachelor), Immature males had significantly lower hair cortisol concentrations than the other groups (p= 0.001). Hair testosterone levels among the four social positions were significantly higher among Stallions (p= 0.04). Hair testosterone concentration was also significantly related to the probability of a male being either a Bachelor or Stallion and was the only variable remaining in AICcmodel selection (p= 0.016, AICc= 32.3, Null AICc= 38.8). While not a significant relationship, Stallions had a negative correlation between hair cortisol concentrations and testosterone (R2= −0.20,p= 0.383), and Bachelors, conversely, had a positive association (R2= 0.43,p= 0.246). Our observations of hormone concentrations in relation to physiological, social, or reproductive parameters in this population suggest trends that are similar to what has been established using blood or other matrices.