The southeastern United States harbors a high diversity of stream fishes, yet little river mileage and few species are protected. Kentucky’s Upper Green River Basin is the subject of a U.S.D.A. Conservation Reserve Enhancement Program. Prior within-subbasin analyses assessing whole assemblage relationships to watershed- and reach-scale environmental variables showed that (a) distributions were due to stream-size gradients and not by land-use patterns or indicators of anthropogenic disturbance, and (b) species were associated either with small, upland segments or only within the largest, deeper segments. Two additional analyses have been performed. First, data was pooled across all subbasins yet divided sites into two distinct watershed groups to assess if there were size-driven biotic responses to environmental variables. Even amongst the smallest stream segments an agricultural influence was not revealed. A series of canonical correspondence analyses (CCA) and indicator species analyses revealed mainly similar stream-size related relationships across the two data groups. Second, data were reduced solely to darter species (Etheostoma and Percina) to reduce noise induced by the large number of species obtained. Similarly, CCA and multiple linear regression models between abundance of significant indicator species analyses and environmental variables similarly revealed only stream size patterns.