Dietary studies on generalist predators may provide valuable information on spatial or temporal changes in the structure of ecological communities. We initiated this study to provide baseline data and determine the utility of stable isotope analysis (SIA) to evaluate the foraging strategies of an opportunistic reptilian predator, the diamondback terrapin (Malaclemys terrapin), which specializes in salt marshes and mangrove estuaries along the Atlantic and Gulf coasts. We evaluated stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values of multiple tissues from terrapins inhabiting mainland and island mangrove habitats in south Florida and potential food sources to examine spatial and temporal variations in terrapin resource use. We fit linear regression models to determine the best predictors of isotopic values for both terrapins and their prey, and Stable Isotope Bayesian Ellipses in R (SIBER) analysis to examine terrapin isotopic niche space and overlap between groups. We identified differences in terrapin isotopic δ¹³C and δ¹⁵N values among all sites. Blood and scute tissues revealed different isotopic compositions and niche overlap between sites, suggesting diets or foraging locations may change over time, and amount of variation is site specific. Niche overlap between size classes was larger for blood (short term) versus scute (long term), suggesting greater variability in food habits or resource isotopes over the long term versus short term. These results demonstrate the usefulness of SIA in examining the spatial and temporal variability in diamondback terrapin resource use within estuary systems and further define their niche within these dynamic food webs.