Hair cortisol is a relatively non-invasive and reliable measure of chronic stress, but it has received limited use, especially in pasture systems. A two-year study was carried out to compare behavioral and physiological (intravaginal temperature, hair, and blood cortisol) responses of ewes (Ovis aries) that grazed black walnut (Juglans nigra) silvopasture (BSP), honeylocust (Gleditsia triacanthos) silvopasture (HSP), or open pastures (OP) treatments. Ewe weights and intravaginal temperatures were recorded once for every 4-week interval. Plasma and hair cortisol concentrations were determined by ELISA. Trail cameras detected animal behavior. Ewe average daily gain was greater in HSP compared with OP (p = 0.0456) but did not differ with BSP (p = 0.4686) across both years. Ewes on OP had higher (p < 0.0001) hair cortisol concentrations than ewes on silvopasture treatments both summers. Ewes on OP had ≥ 0.4 °C higher (p ≤ 0.03) intravaginal temperatures during portions of the afternoon than ewes managed in silvopasture treatments. Ewes on OP spent 500–700% more (p < 0.0001) time standing and 125–150% less (p < 0.0001) time lying down compared with ewes on silvopasture treatments. Hair cortisol measures could be an effective and relatively non-invasive technique for determining long-term chronic stress in grazing animals.