Distinguishing discrete population units among continuously distributed coastal small cetaceans is challenging and crucial to conservation. We evaluated the utility of stable isotopes in assessing group membership in bottlenose dolphins (Tursiops truncatus) off west-central Florida by analyzing carbon, nitrogen, and sulfur isotope values (δ¹³C, δ¹⁵N, and δ³⁴S) of tooth collagen from stranded dolphins. Individuals derived from three putative general population units: Sarasota Bay (SB), nearshore Gulf of Mexico (GULF), and offshore waters (OFF). Animals of known history (SB) served to ground truth the approach against animals of unknown history from the Gulf of Mexico (GULF, OFF). Dolphin groups differed significantly for each isotope. Average δ¹³C values from SB dolphins (−10.6‰) utilizing sea grass ecosystems differed from those of GULF (−11.9‰) and OFF (−11.9‰). Average δ¹⁵N values of GULF (12.7‰) and OFF (13.2‰) were higher than those of SB dolphins (11.9‰), consistent with differences in prey trophic levels. δ³⁴S values showed definitive differences among SB (7.1‰), GULF (11.3‰), and OFF (16.5‰) dolphins. This is the first application of isotopes to population assignment of bottlenose dolphins in the Gulf of Mexico and results suggest that isotopes may provide a powerful tool in the conservation of small cetaceans.