Aquatic turtles represent important biotic components of freshwater ecosystems. The Pecos River watershed is inhabited by six freshwater turtle species, including the widespread Trachemys scripta (Red-eared Slider) and a species of conservation concern, Pseudemys gorzugi (Rio Grande Cooter). Here, we assessed isotopic niche widths of Rio Grande Cooter and niche overlap where it co-occurs with Red-eared Slider in the Pecos River tributaries, New Mexico, USA. We used carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope analyses of two different tissue types: blood and claw. Our results showed niche partitioning among different populations of P. gorzugi and among sex classes within a population. At the sites where both species occur, we documented niche overlap, especially for δ¹⁵N values. Stable isotopes showed similar ellipse area overlap (SEA_B) of T. scripta and P. gorzugi among populations (∼20‰²), but little to no overlap of standard ellipse areas for small sample sizes (SEA_C). The distribution of prey items in the diets of P. gorzugi and T. scripta revealed the differences in resource selection. We observed that differences in the diets of P. gorzugi among populations correspond to local resource availability, suggesting opportunistic foraging behavior of P. gorzugi. Our study aids in understanding the ecology and natural history of P. gorzugi, one of the least studied freshwater turtles in the USA. Moreover, our study provides insights to interspecific relations of T. scripta in their native range.