Movement of invertebrates among large rivers, tributaries, and floodplain lakes or dispersal of adult aquatic insects from riverine or floodplain habitats may provide important subsidies to food webs in receiving habitats. Intensive sampling at habitat interfaces and artificial labelling are two approaches to assess freshwater invertebrate dispersal, but these are difficult to implement at a landscape scale. Natural chemical tracers have been used to track dispersal of fishes and marine invertebrates, but the potential applicability of stable isotope ratios as natural tracers of invertebrate dispersal in freshwater environments has not been assessed. We evaluated stable hydrogen and oxygen isotopes (δD and δ18O) as natural markers of source environment and dispersal of macroinvertebrates in the middle Mississippi River, tributaries and floodplain wetlands. Water and invertebrates were collected from 12 sites during 2007–2008. Water δD and δ18O differed among the river, its tributaries, and floodplain wetlands and were strongly correlated with invertebrate δD and δ18O. Variability in invertebrate δ18O rendered it ineffective as an indicator of invertebrate source environment. Mean δD of Mississippi River invertebrates differed from δD of invertebrates from floodplain wetlands; δD distinguished invertebrates from these two environments with >80% accuracy. Neither δD nor δ18O of aquatic insects changed following emergence from their natal site. Preservation method (ethanol or freezing) did not affect invertebrate δD or δ18O. Invertebrate δD may be a useful natural tracer of natal environment and dispersal in the Mississippi River-floodplain ecosystem and other freshwater systems where spatial variation in water δD is present. Copyright © 2010 John Wiley & Sons, Ltd.