We investigated variation of fish assemblages in response to environmental factors using Long Term Resource Monitoring Program data. Data were collected from 1993 to 2000 from five physical habitats in the unimpounded upper Mississippi River. We captured 89 species composing 18 families. Of these, 26% were fluvial specialists, 25% were fluvial dependent and 49% were generalists. The numerically dominant component of the adult fish assemblage (species accounting for >10% of total catch) accounted for 50% of the assemblage and was comprised of only three species: gizzard shad (Dorosoma cepedianum; 25%), common carp (Cyprinus carpio; 15%) and channel catfish (Ictalurus punctatus; 10%). The dominant component of the YOY fish assemblage was comprised of only two species, which accounted for 76% of the total catch: freshwater drum (Aplodinotus grunniens; 39%) and gizzard shad (37%). We used a cross-validation multivariate approach to explore how adult and young-of-the-year (YOY) assemblages varied with respect to physical habitat and environmental gradients. Furthermore, we were interested how the fish assemblages changed over time. Partial canonical correspondence analyses (pCCA) demonstrated significant effects of physical habitats. Such effects differed between young-of-the-year and adult fishes. The four main environmental gradients influencing overall assemblage structure for both age groups were river elevation, water velocity, conductivity and depth of gear deployment. Morisita's index revealed similar adult assemblage structure over time. However, the YOY assemblage present in 1995 was dissimilar from assemblages present during the other years. We speculate this is a lag effect from the backwater spawning episodes (floodpulse) that occurred with the 500-y flood in 1993. Shannon-Weiner diversity and Camargo's evenness indices were low, but stable across years for the adult assemblage, but varied across years for the YOY assemblage.