Naturally occurring stable isotope and trace elemental markers in otoliths have emerged as powerful tools for determining natal origins and environmental history of fishes in a variety of marine and freshwater environments. However, few studies have examined the applicability of this technique in large river-floodplain ecosystems. This study evaluated otolith microchemistry and stable isotopic composition as tools for determining environmental history of fishes in the Middle Mississippi River, its tributaries, and floodplain lakes in Illinois and Missouri, U.S.A. Fishes were collected from 14 sites and water samples obtained from 16 sites during summer and fall 2006 and spring 2007. Otolith and water samples were analyzed for stable oxygen isotopic composition (δ18O) and concentrations of a suite of trace elements; otoliths were also analyzed for carbon isotopic composition (δ13C). Tributaries, floodplain lakes, and the Mississippi and Lower Missouri Rivers possessed distinct isotopic and elemental signatures that were reflected in fish otoliths. Fish from tributaries on the Missouri and Illinois sides of the middle Mississippi River could also be distinguished from one another by their elemental and isotopic fingerprints. Linear discriminant function analysis of otolith chemical signatures indicated that fish could be classified back to their environment of capture (Mississippi River, floodplain lake, tributary on the Illinois or Missouri side of the Mississippi River, or lower Missouri River) with 71-100% accuracy. This study demonstrates the potential applicability of otolith microchemistry and stable isotope analyses to determine natal origins and describe environmental history of fishes in the Middle Mississippi River, its tributaries, and floodplain lakes. 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 Mississippi River provided by fishes that emigrate from floodplain lakes or tributaries.