Depth reduction is a natural process in floodplain lakes, but in many basins has been accelerated by anthropogenic disturbances. A diverse set of 42 floodplain lakes in the Yazoo River Basin (Mississippi, USA) was examined to test the hypothesis of whether depth reduction was a key determinant of water quality and fish assemblage structure. Single and multiple variable analyses were applied to 10 commonly monitored water variables and 54 fish species. Results showed strong associations between depth and water characteristics, and between depth and fish assemblages. Deep lakes provided less variable environments, clearer water, and a wider range of microhabitats than shallow lakes. The greater environmental stability was reflected by the dominant species in the assemblages, which included a broader representation of large-body species, species less tolerant of extreme water quality, and more predators. Stability in deep lakes was further reflected by reduced among-lake variability in taxa representation. Fish assemblages in shallow lakes were more variable than deep lakes, and commonly dominated by opportunistic species that have early maturity, extended breeding seasons, small adult size, and short lifespan. Depth is a causal factor that drives many physical and chemical variables that contribute to organizing fish assemblages in floodplain lakes. Thus, correlations between fish and water transparency, temperature, oxygen, trophic state, habitat structure, and other environmental descriptors may ultimately be totally or partly regulated by depth. In basins undergoing rapid anthropogenic modifications, local changes forced by depth reductions may be expected to eliminate species available from the regional pool and could have considerable ecological implications.