I assessed fish community data collected by the Oklahoma Conservation Commission from 82 minimally impaired wadeable reference streams in eastern Oklahoma to determine whether existing aquatic ecoregions provide the best framework for spatial classification for the development of biological assessment methods and biocriteria. I used indirect ordination and classification to identify groups of sites that support similar fish communities. Although correspondence was observed between fish assemblages and three montane ecoregions, the classification system must be refined and expanded to include major drainage basins and physical habitat attributes for some areas to adequately partition variance in key measures of biological integrity. Results from canonical correspondence analysis indicated that substrate size and habitat type were the primary physical habitat variables that influenced the fish species composition and community structure.