Flowing water ecosystems often form hierarchical networks, with tributaries combining into successively larger-order streams and rivers. This nested structure can lead to steady, incremental changes in hydrologic attributes such as discharge volume and flood intensity and biological features such as species diversity and population connectivity. Due to regional geologic constraints, however, hydrologic disjunctions may occur, where small-order streams connect directly with large-order rivers. At disjunctions, stream characteristics may change abruptly over short distances, creating discontinuous landscapes for species and populations. We studied the effect of hydrologic disjunctions on aquatic insect communities and populations in a large desert river, the Colorado River in Grand Canyon, where multiple first and second-order streams join directly with the 7th-order mainstem. These disjunctions create differences in mean discharge (<1m3 vs. 273 m3), flood magnitude and timing, water temperature, water chemistry, and sediment profile. We used eDNA to characterize aquatic insect communities and populations at 18 hydrologic disjunctions (a tributary and the adjacent mainstem). We hypothesized that communities would differ between tribs and adjacent mainstem sites. We identified 407 taxa across the entire system, and each trib harbored an average of 53 taxa while mainstem sites contained only 30. Sorensen dissimilarity between tribs and mainstem sites was nearly 70%, with most of this due to turnover (45%) rather than nestedness. Hydrologic discontinuity can impose a limit to dispersal within the river network, and could also affect the ability of the system to respond to future changes in flow and temperature.