Description: Arctic and subarctic environments are among the most inaccessible regions in the world, making biological surveys difficult to conduct. Thus, the insect fauna of these regions has remained inadequately surveyed. The aquatic insect orders Ephemeroptera, Plecoptera, and Trichoptera (EPTs) are particularly abundant and diverse at high latitudes, playing key roles in trophic chains where nutrients are scarce. However, particular aspects of their life cycle make them difficult to study. Specifically, species-level identification requires last-instar larvae or adults which, because they are short lived, are typically not available for all taxa during a particular collecting event. With the initial goal of surveying the biodiversity of these insect groups, we sampled ca.10,000 EPT specimens from 12 locations across northern Canada over two years. Approximately 800 of these were subsequently selected for COI DNA barcoding. Overall, we identified 155 EPT species (58 Ephemeroptera, 41 Plecoptera, 56 Trichoptera) based on a 2% divergence criterion. Compared to other similar studies on EPTs we found higher (particularly among the Plecoptera and Ephemeroptera) and more even diversity, potentially reflecting environmental differences in sampling localities. We further assessed phylogeographic divergence patterns among seven species, finding that eastern and western populations diverged during the Pleistocene Epoch (〈2.5 Ma), with overlapping time frames. This finding highlights the role of potential glacial refugia and subsequent recolonization, as well as the dispersal potential of some EPT species. This study exemplifies how large-scale DNA-based surveys can be combined with phylogeographic inference to better understand the biodiversity and natural history of northern aquatic insect communities.