Fish-habitat associations are poorly known in offshore areas of very large rivers. We examined physical habitat structure and its effect on habitat use and assemblage formation of benthic fishes in the main channel of the Danube River, Hungary. Principal component analysis of physical variables showed that sample unit (i.e., 500 m long reaches) and cross-channel transect-level data of corresponding reaches were highly correlated. We found clear gradients in physical variables from areas with high velocity and higher proportion of hard substratum (pebble and cobble) to areas with low velocity, high mean depth and finer substratum (mainly sand) composition. Variation in velocity was coupled with variation in both mean depth and substratum composition (i.e., Shannon diversity of sediment composition) and higher proportion of silt material. Differences in physical habitat structure (flow, substrate) also manifested among river segments. Classification and regression tree analyses (CART) and fish abundanceoccupancy patterns in the PCA template revealed that many species showed clear responses to environmental heterogeneity (barbel, Barbus barbus; schraetser, Gymnocephalus schraetser; Danube streber, Zingel streber; whitefinned gudgeon, Romanogobio vladikovy; round goby, Neogobius melanostomus) while others (white bream, Blicca bjoerkna) showed very elusive habitat use patterns. Multivariate regression tree analysis confirmed the results of CART and indicated that transectlevel substratum composition was the most important determinant in the formation of benthic assemblages. These results on habitat use can contribute to the more effective conservation management of offshore fish assemblages, which is important due to increasing inland navigation in the Danube River.