Studies in freshwater ecosystems are seeking to improve understanding of carbon flow in food webs and stable isotopes have been influential in this work. However, variation in isotopic values of basal production sources could either be an asset or a hindrance depending on study objectives. We assessed the potential for basin geology and local limnological conditions to predict stable carbon and nitrogen isotope values of six carbon sources at multiple locations in four Neotropical floodplain ecosystems (Paraná, Pantanal, Araguaia, and Amazon). Limnological conditions exhibited greater variation within than among systems. δ15N differed among basins for most carbon sources, but δ13C did not (though high within-basin variability for periphyton, phytoplankton and particulate organic carbon was observed). Although δ13C and δ15N values exhibited significant correlations with some limnological factors within and among basins, those relationships differed among carbon sources. Regression trees for both carbon and nitrogen isotopes for all sources depicted complex and in some cases nested relationships, and only very limited similarity was observed among trees for different carbon sources. Although limnological conditions predicted variation in isotope values of carbon sources, we suggest the resulting models were too complex to enable mathematical corrections of source isotope values among sites based on these parameters. The importance of local conditions in determining variation in source isotope values suggest that isotopes may be useful for examining habitat use, dispersal and patch dynamics within heterogeneous floodplain ecosystems, but spatial variability in isotope values needs to be explicitly considered when testing ecosystem models of carbon flow in these systems.