The utility of an index of biotic integrity (IBI) depends on its ability to distinguish anthropogenic effects on biota amid natural biological variability. To enhance this ability, we examined fish assemblage data from least-disturbed stream sites in Virginia to determine the best way to regionally stratify natural variation in candidate IBI metrics and their scoring criteria. Specifically, we examined metric variation among physiographic regions, U.S. Environmental Protection Agency ecoregions, and drainage basins to judge their utility as regions in which to develop and use distinct versions of the IBI for Virginia warmwater streams. Statewide, metrics differed most among physiographic regions; thus, we recommend their use as IBI regions. Largest differences were found for taxonomic metrics between coastal plain and mountain sites, particularly in numbers of native minnow (Cyprinidae), sunfish (Centrarchidae), and darter (Percidae) species. Trophic and reproductive metrics also differed between coastal plain and more-upland streams, presumably reflecting differences in functional adaptations of fishes to upland versus lowland stream habitats. We suggest three preliminary regional IBis for Virginia, each having a distinctive set of taxonomic, trophic, and reproductive metrics and corresponding scoring criteria.