We examined data collected on fish assemblage structure among three differing floodplain types (broad, moderate, and narrow) during the 1993 flood in the unimpounded reach of the upper Mississippi River. This 500 year flood event provided a unique opportunity to investigate fish-floodplain function because the main river channel is otherwise typically disjunct from approximately 82% of its floodplain by an extensive levee system. Fishes were sampled during three separate periods, and 42 species of adult and young-of-the-year (YOY) fishes were captured. Analysis of similarity (ANOSIM) revealed a significant and distinguishable difference between both adult and YOY assemblage structure among the three floodplain types. Analysis of variance revealed that Secchi transparency, turbidity, water velocity, and dissolved oxygen were significantly different among the floodplain types. However, only depth of gear deployment and Secchi transparency were significantly correlated with adult assemblage structure. None of these variables were significantly correlated with YOY assemblage structure. The numerically abundant families (adult and YOY catches combined) on the floodplain included Centrarchidae, Ictularidae, and Cyprinidae. Both native and non-native fishes were captured on the floodplain, and several of the numerically abundant species that were captured on the floodplain peaked in catch-per-unit-effort 1–3 years after the 1993 flood event. This suggests that some species may have used flooded terrestrial habitat for spawning, feeding, or both. The findings from our study provide much needed insight into fish-floodplain function in a temperate, channelized river system and suggest that lateral connectivity of the main river channel to less degraded reaches of its floodplain should become a management priority not only to maintain faunal biodiversity but also potentially reduce the impacts of non-native species in large river systems.