Concerns about biodiversity and sustainability extend to many ecological systems, including large river systems that are highly modified by human activities. The Mississippi River is one such system that is currently regulated for navigational and flood control purposes, bears a large agricultural nutrient load, and has experienced rapid spread of the exotic zebra mussel, Dreissena polymorpha, since 1991. Human development of the extensive watershed system of the Mississippi River is ongoing and is expected to lead to further changes in river ecology. This study tested whether stable isotope compositions of the zebra mussel could help identify watershed and tributary loading of carbon (C), nitrogen (N), and sulphur (S) to the mainstem river. Zebra mussels were collected seasonally in the lower Mississippi River at Baton Rouge in 1997, and along most of the length of the river in a north–south transect from Minnesota to Louisiana during August 1998. Results showed substantial seasonal variations in C, N and S isotopic compositions of 2‰ or greater, but also that seasonal changes appeared regular and linked to changing watershed inputs and chemistry of the river water. Nitrate was the dominant N nutrient in the Mississippi River, but isotope analyses showed that food webs based on ammonium rather than nitrate were likely important for the zebra mussel. Results from the north–south transect followed expectations based on mixing of mainstem river water with tributaries that had different chemistries, so that local zones of influence were detectable in the combined CNS zebra mussel isotopes downstream of major tributary confluences for the Illinois, Missouri and Ohio Rivers. Overall, the study supports use of stable isotopes to help monitor watershed development and downstream effects on aquatic food webs. Copyright © 2003 John Wiley & Sons, Ltd.