BackgroundFreshwater ecosystems, such as streams, are facing increasing pressures from agricultural land use and recent literature stresses the importance of robust biomonitoring to detect trends in insect decline globally. Aquatic insects and other macroinvertebrates are often used as indicators of ecological condition in freshwater biomonitoring programs; however, these diverse groups can present challenges to morphological identification and coarse-level taxonomic resolution can mask patterns in community composition. Here, we incorporate molecular identification (DNA metabarcoding) into a stream biomonitoring sampling design to explore the diversity and variability of aquatic macroinvertebrate communities at small spatial scales. While individual stream reaches can be very heterogenous, most community ecology studies focus on larger, landscape-level patterns of community composition. A high degree of community variability at the local scale has important implications for both biomonitoring and ecological research, and the incorporation of DNA metabarcoding into local biodiversity assessments will inform future sampling protocols.ResultsWe sampled twenty streams in southern Ontario, Canada, for aquatic macroinvertebrates across multiple time points and assessed local community variability by comparing field replicates taken ten meters apart within the same stream. Using bulk-tissue DNA metabarcoding, we revealed that aquatic macroinvertebrate communities are highly diverse at small spatial scales with unprecedented levels of local taxonomic turnover. We detected over 1600 Operational Taxonomic Units (OTUs) from 149 families, and a single insect family, the Chironomidae, contained over one third of the total number of OTUs detected in our study. Benthic communities were largely comprised of rare taxa detected only once per stream despite multiple biological replicates (24–94% rare taxa per site). In addition to numerous rare taxa, our species pool estimates indicated that there was a large proportion of taxa that remained undetected by our sampling regime (14–94% per site). Our sites were located across a gradient of agricultural activity, and while we predicted that increased land use would homogenize benthic communities, this was not supported as within-stream dissimilarity was unrelated to land use. Within-stream dissimilarity estimates were consistently high for all levels of taxonomic resolution (invertebrate families, invertebrate OTUs, chironomid OTUs), indicating stream communities are very dissimilar at small spatial scales.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12862-023-02118-w.