We sampled fish assemblages in 41 floodplain lakes in the Ohio River Basin in the summer of 2012. We collected 2427 individual fishes in 70 species. Mean abundance of individuals at sites was 66, and mean species richness per site was 8.1. We used two multivariate procedures to predict fish-assemblage variation from habitat and environmental variables: an indirect gradient approach (reciprocal averaging [RA]) and a direct gradient approach (canonical correspondence analysis [CCA]). When we applied a forward selection process in the CCA, the habitat and environmental variables that contributed significantly to explaining variation in fishes were mean elevation, latitude, maximum depth, conductivity, longitude, dissolved oxygen, cobble and sand substrates, and lake-surface area. RA provided different results that suggested the presence of additional environmental gradients we did not quantify. Our results show that floodplain lakes in the Ohio River basin contain high species richness and are important habitats to conserve because they have the potential to act as source pools for river fish populations.