Stable 13C, 15N, and 34S isotopes were analyzed to define the feeding habitats of Acipenser oxyrinchus desotoi in the Suwannee and Yellow River populations. For the majority (93.9%) of Suwannee subadults and adults, 13C and 34S signatures indicate use of nearshore marine waters as primary winter feeding habitat, probably due to the limiting size of the Suwannee Sound estuary. In the Yellow River population, 13C isotope signatures indicate that adults remain primarily within Pensacola Bay estuary to feed in winter, rather than emigrating to the open Gulf of Mexico. A minor Suwannee River subset (6% of samples), comprised of juveniles and subadults, displayed 13C signatures indicating continued feeding in freshwater during the spring immigration and fall emigration periods. This cannot be interpreted as incidental feeding since it resulted in a 20.5% turnover in tissue δ13C signatures over a 1–3 month period. Cessation of feeding in the general population does not coincide with high river water temperatures. The hypothesis of reduced feeding in freshwater due to localized prey depletion as a result of spatial activity restriction is not supported by the present study. Instead, Suwannee River A. o. desotoi appear to follow two trophic alternatives; 1) complete cessation of feeding immediately upon immigration in spring, continuing through emigration 8–9 months later (the predominant alternative); 2) continued intensive feeding for 1–3 months following immigration, switching to freshwater prey, selected primarily from high trophic levels (i.e., large prey). Stable –34S data verifies that recently immigrated, fully-anadromous A. o. desotoi adults had fed in nearshore marine waters, not offshore waters.