An understanding of the interactions among local environmental factors (e.g., physical habitat and water quality) and aquatic assemblages is essential to conserve biodiversity in tropical and subtropical headwater streams. We evaluated the relative importance of multiple physical and chemical habitat variables that influence the richness of Ephemeroptera, Plecoptera, and Trichoptera (EPT) assemblages in wadeable Brazilian Cerrado (savanna) streams. We sampled macroinvertebrate assemblages and quantified physical and chemical habitat in 79 randomly selected sites in 2 Cerrado basins in southeastern Brazil. The environmental variables selected by multiple regression models (MLRs) via corrected Akaike Information Criteria (AICc) contributed significantly to variation in EPT taxon richness. The variance explained by physical-habitat variables was slightly greater in the Upper São Francisco Basin (adjusted R² = 0.53) than in the Upper Araguari Basin (adjusted R² = 0.46), and both were greater than the variance explained by a combined basin model (adjusted R² = 0.39). Physical-habitat variables were more important than water-quality variables in structuring EPT genera in streams with catchments dominated by agriculture or pasture land uses. Regional models can be improved by incorporating basin-specific information to refine biological assessments and to provide better understanding of the interactions that maintain biodiversity in stream networks.