Under the EU Water Framework Directive, ecological assessment and management are based on type-specific reference conditions. In the EU it may be difficult to find sites in large rivers with at least near-natural conditions, though this is not the case in southeast Europe, where stretches of large rivers still exist with at least near-natural conditions, meaning that there is little or no disturbance from hydromorphological alteration, water quality, land use in the catchment and alien species. We examined benthic invertebrate assemblages in 45 samples collected from near-natural sites of several large rivers: Sava, Drava, Mura, Kupa and Una. The near-natural benthic invertebrate assemblages of large rivers contained several rare or remarkable species, especially among stoneflies, e.g., Marthamea vitripennis, Xanthoperla apicalis. We compared benthic invertebrate communities in river sections with fine and coarse substrates and in three eco-hydromorphological (ECO-HM) types of large rivers, reflecting habitat heterogeneity: lowland-deep, lowland-braided and intermountain. Multivariate analysis of variance (PERMANOVA) was used to statistically evaluate similarities among assemblages. It was found that the composition of benthic invertebrate assemblages varied by both ECO-HM types and substrate category. Similarity percentage (SIMPER) analysis showed that the average dissimilarity of benthic invertebrate assemblages was high between all ECO-HM type pairs and between fine and coarse substrate. We found that habitat heterogeneity and substrate independently influenced benthic invertebrate assemblages. To achieve ecological goals in the management of large rivers, in addition to functionality, a holistic view with at least near-natural assemblages, including the names of the taxa present, should also be considered.