Identifying naturally-regulated spatial and temporal variations of benthic macroinvertebrates is critical to effective assessment and conservation of aquatic ecosystems, but little is known about these variations in large rivers in East Asian monsoon region. Here, we address this issue by measuring within-one-year longitudinal and seasonal variability in macroinvertebrate assemblages in such a broadly natural river across its whole watershed. Along longitudinal gradients, taxon richness, Shannon Wiener diversity and mayflies and caddisflies significantly decreased downstream but chironomids and oligochaeta increased. Taxon richness, diversity indices, and abundance of most taxa all had lowest value in summer. Assemblage structure showed both significant but larger longitudinal than seasonal variations, with a clear separation of upstream and most midstream sites from downstream ones in ordination plot. Different environmental and spatial variables were significant for distinguishing macroinvertebrate assemblages among four seasons, although substrate and PCNM1 emerged as important in all seasons. Variance partitioning analyses indicated stronger environmental control than spatial structuring of community composition in all seasons, with pure environmental factors explaining most community variation. These observed patterns contribute to understanding of sources of uncertainty in bioassessment and thus have implications for ecological monitoring and assessment using macroinvertebrates in rivers in monsoon regions. (C) 2016 Elsevier Ltd and INQUA. All rights reserved.