Resource use and niche partitioning has rarely been shown in ancient C₃-dominated environments through analysis of stable isotope values, even though before 7 million years ago worldwide environments were predominantly composed of C₃ plant taxa. This study explores whether the analysis of stable carbon isotopes from ungulate tissues can accurately identify resource use and niche partitioning in a modern C₃-dominated ecosystem in North America, in order to lay the groundwork for application to more ancient ecosystems. δ¹³C values were obtained from the scat, collagen, and tooth enamel from populations of Antilocapra americana, Bison bison, Cervus elaphus, Odocoileus hemionus, and Ovis canadensis in Yellowstone National Park, and used to trace isotopic uptake and incorporation from the diet into herbivore hard tissues. Significant differences were observed among taxa in δ¹³C values from all three of the sample materials. Scrutinizing the carbon isotope values further showed that elk and mule deer had the widest range in isotopic values, implying the widest range of resource use. Similarities among taxa in the δ¹³C of scat concur with other studies that attribute the similarities to winter habitat use. Because (1) significant differences were observed in the δ¹³C values of scat, collagen and ultimately tooth enamel and (2) the isotopic inputs of carbon are relatively well understood, and (3) carbon isotope values can be obtained from non-altered fossil material, this methodology appears practicable for objectively examining ecological relationships, such as resource use and niche partitioning, among taxa in ancient C₃-dominated ecosystems of North America and elsewhere, and suggests the technique will be valuable in investigating resource use and niche partitioning among taxa prior to the C₄ global carbon shift.