DNA barcoding (using a standardized sequence of the mitochondrial CO1 gene) was used to determine the aquatic insect species richness of two sites along White Clay Creek in Pennsylvania. Water quality assessment at the sites did not change from good (14.6, previous MAIS score 13.2) and fair (9.4, earlier MAIS 7.3), but barcoding increased the species richness and provided a much more detailed analysis by detecting cryptic species. Aquatic insect identifications by an amateur biologist and by expert taxonomists using traditional methods based on morphology were compared to DNA barcoding. The amateur biologist’s identifications were limited to order and family while expert taxonomists were able to identify 44 different species and DNA barcoding indicated 128 different species. 84% of the 1786 specimens that were submitted for barcoding generated a successful DNA sequence. DNA barcoding revealed the presence of more species than expert taxonomists identified as shown in the following listing of insect orders with comparison of numbers of species identified by expert taxonomists and DNA barcoding: Diptera (23 expert spp. and 128 barcoding spp.), Ephemeroptera (6 expert spp. and 16 barcoding spp.), Plecoptera (0 expert spp. and 6 barcoding spp), Trichoptera (9 expert spp. and 14 barcoding spp), and Coleoptera (6 expert spp. and 6 barcoding spp). Station 12 had an overall higher species richness and abundance of Chironomidae; Chironomids accounted for 63% of the specimens with 64 species. Chironomids made up only 30% of the specimens at Station 11 and EPT richness was higher. The increase in the abundance and species richness of Chironomidae at Station 12 supported the previous findings of a lower water quality than that occurring at Station 11. Barcoding, when combined with traditional aquatic macroinvertebrate sampling, provides the most accurate and cost effective method to determine the water quality of fresh water ecosystems.