Estuarine habitat occupied by Alligator mississippiensis, a primarily freshwater species, is spatially and temporally heterogeneous largely due to a salinity gradient that fluctuates. Using long-term night light survey data, we examined seasonal patterns in alligators’ habitat use by size classes in midstream and downstream estuary zones of Shark River, Everglades National Park, in southern Florida. We observed predominantly large-sized alligators (total length ≥ 1.75 m); observations of alligators in the small size classes (0.5 m ≤ total length < 1.25 m) were rare especially in the higher-salinity downstream zone. The density of alligators in the downstream zone was lower than that of the midstream zone during the dry season when salinity increases due to reduced precipitation. Conversely, the density of the large size alligators was higher in the downstream zone than in the midstream zone during the wet season, likely because of reduced salinity. We also found a significant declining trend over time in the number of alligators in the dry season, which coincides with the reported decline in alligator relative density in southern Florida freshwater wetlands. Our results indicated high adaptability of alligators to the fluctuating habitat conditions. Use of estuaries by alligators is likely driven in part by physiology and possibly by reproductive cycle, and our results supported their opportunistic use of estuary habitat and ontogenetic niche shifts.