Elevation gradients allow scientists to observe changes in fauna over a range of abiotic conditions. A variety of trends in aquatic insect diversity patterns across elevation have been reported. However, many of these studies are confounded because they include streams at lower elevations, which are often larger in size and more polluted than their higher-elevation counterparts. Moreover, such studies always relied solely on morphological delineation of taxa, thereby potentially overlooking cryptic diversity. We reduced these limitations by sampling only minimally impacted wadeable streams across an elevation gradient and by combining morphological taxonomy with deoxyribonucleic acid (DNA) barcoding to identify taxa. We collected numerically abundant Ephemeroptera, Plecoptera, and Trichoptera (EPT) from single streams at ∼200-m elevation intervals across >1000-m transects in 3 watersheds draining the eastern slope of the Colorado Rocky Mountains. Based on morphology alone, we identified 49 numerically abundant EPT morphospecies across 26 sites. Using DNA barcoding, we found 69 distinct lineages that probably represent distinct species. EPT species richness was highest at midelevations, and rates of turnover along elevation transects showed no consistent elevation trend or trend among ecological zones defined by vegetation. >-diversity across sites at comparable elevations in different watersheds showed a negative trend with increasing elevation that was marginally significant for DNA barcode taxa (p = 0.051) but not for morphospecies. Furthermore, significant (p < 0.05) differences in taxon richness, turnover, and lateral >-diversity values generated by DNA barcoding underscore the ability of molecular tools to quantify patterns in aquatic insect diversity across elevations.