The endangered black-footed ferret (Mustela nigripis) has been the focus of intensive captive breeding and reintroduction projects for several decades. To better understand nutritional provision during captivity, primary prey items (prairie dogs) of free-ranging black-footed ferret populations were sampled from 6 native habitat sites in Wyoming and Colorado over a one-year period. Morphometrics and nutritional analyses including proximate composition (water, crude fat, crude protein, ash), vitamins A and E, and select macro-and microminerals were conducted on black-tailed (BT, Cynomys ludovicianus, n = 81) and white-tailed (WT, C. leucurus; n = 58) prairie dogs. Stomach and intestinal contents were extracted and sampled separately from other carcass components. Multivariate linear modeling of data was used to determine the influence of environmental (season, site) and prey-based (species, age, sex) factors on prey nutritional composition. Seasonality impacted the nutrient profiles of prairie dogs as food for black-footed ferrets, affecting carcass, stomach, and intestinal samples in most nutrients evaluated for both species. Carcass and subcutaneous fat concentrations were lowest in spring for both species compared with other seasons. Conversely, fat-soluble vitamin A in carcasses was highest in the spring for both species. Vitamin E was also highest in the spring for WT, but highest in the winter for BT, although no comparative winter data were available for the hibernating WT. Macronutrient composition did not differ between sexes for WT, but carcass fat was higher, hence protein lower, in female vs male BT. Age class and site-specific differences detected for some nutrients suggested possible underlying feeding ecology differences. Given the ongoing concerns regarding ex situ population sustainability and the documented role of nutrition in black-footed ferret health and reproduction, these seasonal nutrient profiles provide valuable guidelines for optimizing managed feeding programs for this endangered species, and similar considerations in prey nutrient variability can be applied to feeding programs of other carnivorous species.