Stream site classification is a fundamental step in defining reference conditions for freshwater bioassessments globally. Landscape regionalisation and stream typology approaches have both been used to determine classes that reduce inherent environmental variation and to show classes with ecological meaning. We evaluated the applicability of ecoregion and stream typology approaches in a river basin located between the Neotropical Savanna and the Atlantic Forest biomes in south-eastern Brazil. We delimited two ecoregions and assessed whether stream types within each ecoregion would further reduce environmental and macroinvertebrate assemblage variability. In addition, we determined indicator taxa for each of the reference condition classes. Our results confirm the importance of defining ecoregions a priori and for using a nested a posteriori stream typology approach for further explaining macroinvertebrate assemblage variation. Geology and natural vegetation physiognomy were the key ecoregion factors likely to influence macroinvertebrate assemblages, and stream dimension attributes best delimited meaningful stream types. Although stream classification is a very important step in any monitoring, management or restoration program, it has been widely neglected in many tropical regions. In this study we demonstrated how it can be conducted to determine macroinvertebrate assemblage potentials even with a relatively limited number of sites.