Hydrologic alterations within the Everglades have degraded American alligator (Alligator mississippiensis) habitat, reduced prey base, and increased physiological stress. Alligator body condition declined across many management areas from 2000 through 2014, prompting us to investigate the relationship between their intraspecific isotopic niche dynamics and body condition. Alligators within the estuary had a larger niche driven by a wider range in stable carbon isotope ratios than those sampled in freshwater habitats. Spatially, model predictability was higher at the smaller scale, reflecting the variability in basal sources and biochemistry among capture sites. Male niches were often larger than those of females, driven by wider ranges of δ¹³C values, suggesting that they differ in their proportional use of habitats and or resources. However, the similar ranges of δ¹⁵N values indicated both sexes foraged within the same trophic level. Furthermore, while not significantly different, large alligators often had a larger niche with elevated δ¹⁵N values compared to medium-sized alligators. Although alligators utilize similar stable carbon and nitrogen isotope pools through time, there was considerable temporal variability. These temporal variations in alligators’ isotopic niche were likely influenced by seasonal hydrologic fluctuations within each site, with their niches often being larger in the spring captures than the fall captures. Alligators’ body condition estimates were correlated with intraspecific niche characteristics, including the mean centroid distance between sexes and the interaction between male and female niche size and overlap, within a site, capture period, and year. The variability in intraspecific niche dynamics, landscape heterogeneity, and dynamic hydrology are considerations for designing sustainable management strategies to conserve and enhance alligator populations within the Everglades landscape.