Stable isotope composition of the eastern oyster Crassostrea virginica was characterized from three estuaries in the Ten Thousand Islands (Florida). Freshwater in flow from watershed management is affecting this region and has deeply modified the salinity gradient. Stable carbon and nitrogen isotopes were used to trace specific sources of organic matter and the influence of these sources of carbon on the diet of the eastern oyster in the Greater Everglades. Oysters, as well as particulate organic matter (POM) and benthicmicroalgae (BMA) were sampled in three bays at three different stations from upstream to downstream during both wet and dry seasons. Significant salinity differences were observed in all estuaries between summer (wet) and winter (dry) seasons and were linked to freshwater inputs into the bays. Temperature followed a typical seasonal trend. Oysters were enriched in δ¹⁵N compared with POM and BMA, and similarly or slightly enriched in δ¹³C for both seasons. Lighter δ¹³C values in the upper stations in the estuaries suggest input of organic matter from terrestrial sources. Stable isotopes showed that oysters fed more on POM than on BMA. Condition index of oysters varied between stations and seasons in the three estuaries. Because the quality of organic matter consumed by oysters ultimately impacts the health of the oyster, changes in water quality and quantity of freshwater entering estuaries will have implications for future management of the habitat for this ecologically and economically important species.