We sampled Eurycea pterophila, typically considered a spring-associated species, from unprecedented depths within Jacob's Well, a natural cave in the Trinity Aquifer. Nearly all of the captures followed high spring-flow events (25–70 cubic feet per second [cfs]), with almost no individuals seen or trapped during low flow (<10 cfs). We analyzed the cytochrome b mitochondrial gene DNA sequences and identified four haplotypes present at Jacob's Well, two of which were unique to the site. However, this variation fit within the range of genetic variation for E. pterophila, indicating there is only one species present. Comparisons with other central Texas species indicate this population is part of the previously reported polytomy referred to as the Eurycea neotenes complex. Because our study had a large sample size from a single locality, we were able to detect discord between our phylogenetic tree, which showed uniformity among individuals from Jacob's Well and nearby populations, and our F_ST analyses, which showed no migrants among these populations. These observations highlight the need for larger sample sizes to illuminate relationships among central Texas Eurycea populations.