Macroinvertebrates are widely used as bio-indicators in streams and rivers, and it is usually assumed that their community composition is primarily controlled by local environmental conditions. We examined the distribution of macroinvertebrates within the Guadalupe River basin (3256 km2) in Central Texas across physiographic gradients. Spatial analysis with variables that considers flow direction, connectivity and distances between sites (asymmetric eigenvector maps, AEM) detected distinctive communities in the lower reaches of the mainstem, in spring-influenced reaches, and in a tributary with intermittent reaches. Variation partitioning with redundancy analysis showed that large-scale factors, i.e. riverine network patterns (large-scale AEM variables), climatic variation and ecoregion explained a significant proportion (28%) of the variation in community composition within a river basin. The riverine network patterns were the most important factor, explaining 12% alone. Local environmental factors were significant, but completely confounded within these spatial patterns. We propose that there are distinctive macroinvertebrate communities depending on the location in the river network and this may apply to other (subtropical) rivers, which should be tested by future studies. We recommend spatial analysis that considers distances and connectivity within a river network as a powerful tool to recognize multiscale riverine network patterns, which can help to identify priority areas for conservation and to develop sound monitoring programs.