Around 1000 obligate cave species have been described from the continental United States. This taxonomically diverse group of species contains both terrestrial obligate cave species (troglobites) and aquatic obligate cave species (stygobites). The greatest diversity of troglobites in the United States occurs on the southern Cumberland Plateau in south-central Tennessee and northeastern Alabama. The troglobitic spider Nesticus barri Gertsch 1984 is known from nearly 60 caves in this area. We studied the mitochondrial phylogeographic structuring of this species, sampling individuals from twelve caves across the species' range. We found that N. barri populations within individual caves are generally not genetically diverse; that N. barri is divided into genetically distinct subpopulations, with mitochondrial cytochrome oxidase I genetic distances between subpopulations ranging from 0.021 to 0.045; and that female-based migration between caves is minimal or nonexistent, even over small geographic scales (< 15 km). This is the first genetic study of a troglobitic taxon from this biodiverse region. Our results contrast with those from previous studies on stygobitic crayfish from this area, which showed high levels of gene flow between caves.