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abstract: Initiating a breeding program using animals sourced from the wild can be challenging due to the time it takes to determine their basic reproductive traits in the ex situ environment. Often, the needs for such a program arise rapidly due to a catastrophic event. After multiple wildfires decimated much of the endangered Mt. Graham red squirrel’s (Tamiasciurus fremonti grahamensis) limited habitat, the U.S. Fish and Wildlife Service and the Phoenix Zoo (Phoenix, Arizona, USA) initiated a pilot breeding program using wild-sourced individuals, including the five (three males, two females) studied here. To inform the breeding program, we sought to characterize the reproductive biology of the Mt. Graham red squirrel by developing non-invasive and/or or minimally invasive methods. Our specific objectives were to: 1) characterize reproductive biology via fecal hormone metabolite analysis; 2) determine how reproductive hormones vary across the breeding season; 3) determine the relationship between the hormonal analysis and the physiological changes, and 4) identify a non-invasive or minimally invasive method to rapidly assess when females are close to ovulation. Results showed that follicular phases (number of days fecal estrogen metabolites (FEMs) were elevated) lasted ~26 days (range, 14–51 days). The only female to ovulate ovulated three times with fecal progestogen metabolites (FPMs) remaining elevated for ~21 days. Peak vulvar swelling in this female was associated with higher FEM and the peri-ovulatory period, but vaginal cytology results were less useful for predicting ovulation. For males, scrotum enlargement occurred in January and February and fecal androgen metabolites (FAMs) were elevated in March and June, with lowest concentrations in August, October, and December. Although no successful copulation was observed among paired squirrels, this study provides valuable information about Mt. Graham red squirrel reproductive biology and establishes non-invasive methods that can be used to determine reproductive readiness and make ex situ management decisions. Furthermore, this ex situ study is the first to characterize estrogen and progesterone metabolite patterns for an American tree squirrel (Tribe: Sciurini) across the breeding season.