Responses of macroinvertebrate communities to human pressure are poorly known in large rivers compared with wadeable streams, in part because of variable substrate composition and the need to disentangle pressure responses from underlying natural environmental variation. To investigate the interaction between these factors, we sampled macroinvertebrates from the following: (i) submerged wood; (ii) littoral substrates < 0.8 m deep; and (iii) inorganic substrates in deep water (> 1.5 m) benthic habitats in eleven 6th- or 7th-order New Zealand rivers spanning a catchment vegetation land cover gradient. Cluster analysis identified primary site groupings reflecting regional environmental characteristics and secondary groupings for moderate gradient rivers reflecting the extent of catchment native vegetation cover. Low pressure sites with high levels of native vegetation had higher habitat quality and higher percentages of several Ephemeroptera and Trichoptera taxa than sites in developed catchments, whereas developed sites were more typically dominated by Diptera, Mollusca and other Trichoptera. Partial regression analysis indicated that the combination of underlying environment and human pressure accounted for 77–89% of the variation in Ephemeroptera, Trichoptera and Plecoptera taxa richness, %Diptera and %Mollusca, with human pressure explaining more variance than underlying environment for %Mollusca. Analysis of replicate deepwater and littoral samples from moderate gradient sites at the upper and lower ends of the pressure gradient indicated that total Trichoptera and Diptera richness and %Diptera responded to land use differences in these boatable river catchments. Responses to human pressure were substrate specific with the combination of littoral and deepwater substrates providing the most consistent response and yielding the highest number of taxa. These results indicate that multiple substrate sampling is required to document the biodiversity and condition of boatable river macroinvertebrate communities and that spatial variation in the underlying natural environment needs to be accounted for when interpreting pressure–response relationships.