1. Aquatic habitats and biotic assemblages in subalpine headwaters are sensitive to climate and human impacts. Understanding biotic responses to such perturbations and the contribution of high-elevation headwaters to riverine biodiversity requires the assessment of assemblage composition among habitat types. We compared aquatic insect assemblages among headwater stream segment types in relict glaciated subalpine basins in Mt. Rainier National Park, Washington, USA. 2. Aquatic insects were collected during summer and autumn in three headwater basins. In each basin, three different stream segment types were sampled: colluvial groundwater sources, alluvial lake inlets, and cascade-bedrock lake outlets. Ward's hierarchical cluster analysis revealed high β diversity in aquatic insect assemblages, and non-metric multidimensional scaling indicated that spatial and temporal patterns in assemblage composition differed among headwater stream segment types. Aquatic insect assemblages showed more fidelity to stream segment types than to individual basins, and the principal environmental variables associated with assemblage structure were temperature and substrate. 3. Indicator species analyses identified specific aquatic insects associated with each stream segment type. Several rare and potentially endemic aquatic insect taxa were present, including the recently described species, Lednia borealis (Baumann and Kondratieff). 4. Our results indicate that aquatic insect assemblages in relict glaciated subalpine headwaters were strongly differentiated among stream segment types. These results illustrate the contribution of headwaters to riverine biodiversity and emphasise the importance of these habitats for monitoring biotic responses to climate change. Monitoring biotic assemblages in high-elevation headwaters is needed to prevent the potential loss of unique and sensitive biota.