The Prairie Pothole Region’s myriad wetlands and small lakes contribute to its stature as the “duck factory” of North America. The fishless nature of the region’s aquatic habitats, a result of frequent drying, freezing, and high salinity, influences its importance to waterfowl. Recent precipitation increases have resulted in higher water levels and wetland/lake freshening. In 2012–13, we sampled chemical characteristics and vertebrates (fish and salamanders) of 162 Prairie Pothole wetlands and small lakes. We used non-metric multidimensional scaling, principal component analysis, and bootstrapping techniques to reveal relationships. We found fish present in a majority of sites (84 %). Fish responses to water chemistry varied by species. Fathead minnows (Pimephales promelas) and brook sticklebacks (Culaea inconstans) occurred across the broadest range of conditions. Yellow perch (Perca flavescens) occurred in a smaller, chemically defined, subset. Iowa darters (Etheostoma exile) were restricted to the narrowest range of conditions. Tiger salamanders (Ambystoma mavortium) rarely occurred in lakes with fish. We also compared our chemical data to similar data collected in 1966–1976 to explore factors contributing to the expansion of fish into previously fishless sites. Our work contributes to a better understanding of relationships between aquatic biota and climate-induced changes in this ecologically important area.