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.