This dataset contains single nucleotide polymorphism (SNP) information for aquatic insect species collected in tributaries of the Colorado River in Grand Canyon (Arizona, USA), as well as SNP information for individuals collected from reference reaches of the Upper Colorado River Basin in Utah. This dataset focuses specifically on three species that were common and widely distributed throughout tributary streams in Grand Canyon: a mayfly (Fallceon quilleri), a caddisfly (Hydrospyche oslari), and a water strider (Rhagovelia distincta).