DNA barcoding has revealed unrecognized species in several animal groups. In this study we have employed DNA barcoding to examine Hyalella, a taxonomically difficult genus of amphipod crustaceans, from sites in the southern Great Basin of California and Nevada, USA. We assessed the extent of species diversity using a species screening threshold (SST) set at 10 times the average intrapopulation cytochrome c oxidase subunit I (COI) haplotype divergence. Despite the fact that this threshold approach is more conservative in delineating provisional species than the phylogenetic species concept, our analyses revealed extraordinary levels of cryptic diversity and endemism. The SST discriminated two provisional species within Hyalella sandra, and 33 provisional species within Hyalella azteca. COI nucleotide divergences among these provisional species ranged from 4.4% to 29.9%. These results have important implications for the conservation of life in desert springs — habitats that are threatened as a result of groundwater over-exploitation.