Percina aurolineata (Percidae: subgenus Hadropterus) (Goldline Darter) is a federally threatened species that currently exists in disjunct populations in the Coosawattee River, GA, and Cahaba River, AL. These 2 Mobile Basin drainages are home to considerable endemism, and these disjunctions may actually represent cryptic diversity within Goldline Darter. We examined sequence data from the mitochondrial cytochrome b gene (cyt b) and nuclear recombination-activation gene exon 1 (RAG1) from specimens (n = 34) collected from 4 streams in the Coosawattee River drainage and 4 streams in the Cahaba River drainage for the purpose of assessing phylogenetic structure and genetic divergence to test the hypothesis that the disjunct populations of Goldline Darter represent a single species. Specimens from each of the rivers sampled were not resolved as a clade in any analysis. For cyt b, divergence within the Coosawattee was 0.8%, divergence within the Cahaba was 0.3%, and net divergence between populations was 0.4%. For RAG1, divergence within the Coosawattee was 0.0%, divergence within the Cahaba was 0.1%, and net divergence between populations was 0.0%. We detected a unique allele for RAG1 with a frequency of 0.559 in the Cahaba specimens. No clades were resolved that contained specimens representative of only one locality and the difference between mean divergence among and within rivers was low; thus, these results support the hypothesis that the disjunct populations of Goldline Darter represent a single species and an evolutionarily significant unit. The divergence of allele frequencies among Cahaba and Coosawattee for RAG1 qualifies them as separate management units, and future conservation efforts should manage them as such.