Knowledge of habitats used by fish throughout their life history is important for management and conservation of riverine fish populations and habitats. Naturally occurring chemical markers in otoliths have recently been used to determine natal origins and environmental history of fishes in a variety of marine and freshwater environments. However, to our knowledge no studies have examined the applicability of this technique in large floodplain rivers in the U.S.A. We evaluated otolith microchemistry and stable isotopic composition as tools for determining origins of fishes in the lower Illinois River, its tributaries, and floodplain lakes. Fishes were collected from eight sites during summer 2006 and two additional sites in spring 2007. Water samples were obtained from these 10 sites plus one additional tributary during summer and fall 2006 and spring 2007. Otolith and water samples were analyzed for δ18O and a suite of trace elements; otoliths were also analyzed for δ13C. Tributaries, floodplain lakes, and the Illinois River possessed distinct isotopic and elemental signatures, principally driven by differences in δ18O and δ13C among floodplain lakes, the Illinois River, and tributary streams. Otoliths reflected differences in water chemistry among habitats. Relationships between water and otolith δ18O and Sr:Ca were not significantly different among species, but some differences in relationships between water and otolith Ba:Ca among species were detected. Linear discriminant function analysis with a leave-one-out jackknife procedure on otolith δ18O and δ13C indicated that fish may be classified back to environment (Illinois River, tributary, or floodplain lake) of capture with 80-98% accuracy. Otolith microchemistry and stable isotope analyses provide a potentially effective means for determining recruitment sources and environmental history of fishes in the Illinois River. The ability to reconstruct environmental history of individual fish using naturally occurring isotopic markers in otoliths may also facilitate efforts to quantify nutrient and energy subsidies to the Illinois River provided by fishes that emigrate from floodplain lakes or tributaries.