The exceptional groundwater community inhabiting the karstic Edwards-Trinity Aquifer system in central Texas has inspired generations of biologists seeking to understand diversification in an extreme environment. Since the late 1990s, molecular genetic tools have increasingly been used to uncover hidden diversity and infer the evolutionary history of groundwater species inhabiting the Edwards-Trinity system. The field of phylogeography—the study of the spatial distribution of genealogical lineages within and among intraspecific populations and closely related species—has provided unparalleled insight into patterns of Edwards-Trinity groundwater biodiversity. Similar to other global groundwater biodiversity hotspots, phylogeographic studies in the Edwards-Trinity Aquifer system have documented exceptionally high levels of endemism and strong population structure due to isolation across naturally fragmented habitat. Cryptic species (two or more morphologically similar but genetically distinct species) have been discovered in a number of phylogeographic investigations, including Eurycea salamanders, Dionda minnows, and Stygobromus amphipods. A number of these species are threatened or endangered with extinction due to habitat loss and degradation resulting from urbanization. Accurately delimiting species boundaries has had significant implications for biodiversity and groundwater conservation in the Edwards-Trinity region because the Endangered Species Act has been used to regulate unrestricted groundwater withdrawal in the eastern Edwards Aquifer where listed species are found. New developments in deoxyribonucleic acid (DNA) sequencing technology coupled with advancements in model-based inference will provide powerful tools for furthering our understanding of Edwards-Trinity biodiversity and predicting its response to a rapidly changing environment.