Sympatric species are expected to differ in resource use in order to avoid competition, leading to niche partitioning in natural communities. Invasive species can disrupt community structure, but these impacts are often difficult to measure. In this study, we used stable isotope analysis of carbon and nitrogen to investigate dietary niche partitioning of freshwater turtle assemblages at three sites in the Middle Rio Grande Basin of New Mexico. We sampled four turtle species common to the region, including Painted Turtles (Chrysemys picta), Spiny Softshells (Apalone spinifera), Common Snapping Turtles (Chelydra serpentina), and invasive Red-Eared Sliders (Trachemys scripta elegans). We found that species composition at the study site with the most anthropogenic influence, the Rio Grande Nature Center in the urban population center of Albuquerque, was dominated by invasive T. s. elegans, while study sites in more remote areas (Sevilleta and Bosque del Apache National Wildlife Refuges) primarily consisted of native species. Furthermore, we observed high overlap in niche space among turtle species at the Rio Grande Nature Center, but evidence consistent with niche partitioning at the other two sites. We found higher nitrogen levels in the more carnivorous A. spinifera and C. serpentina compared to other species. We also found evidence for an ontogenetic shift toward a less carnivorous diet in T. s. elegans. Our study provides valuable baseline information about understudied turtle assemblages within the Middle Rio Grande region and suggests there is potential for competition between invasive T. s. elegans and native turtle species in New Mexico.