Effects of local environmental influences on the structure of fish assemblages were evaluated from 159 sites in two regions of the Great Plains with limited anthropogenic disturbance. These regions offered an opportunity to evaluate the structure and variation of streams and fish assemblages within the Great Plains. We used canonical correspondence analyses to determine the influence of environmental conditions on species abundances, species occurrences and assemblage characteristics. Analysis of regions separately indicated that similar environmental factors structured streams and fish assemblages, despite differences in environmental conditions and species composition between regions. Variance in fish abundance and assemblage characteristics from both regions was best explained by metrics of stream size and associated metrics (width, depth, conductivity and instream cover). Our results provide a framework and reference for conditions and assemblage structure in North American prairie streams.