Cave beetles of the eastern USA are one of many poorly studied groups of insects and nearly all previous work delimiting species is based solely on morphology. This study assesses genetic diversity in the monotypic cave carabid beetle genus Darlingtonea Valentine 1952, to test the relationship between putative geographical barriers to subterranean dispersal and the boundaries of genetically distinct groups. Approximately 400bp of the mitochondrial cytochrome oxidase I (COI) gene was sequenced from up to four individuals from each of 27 populations, sampled from caves along the escarpments of the Mississippian and Cumberland plateaus in eastern Kentucky, USA. The 81 individuals sequenced yielded 28 unique haplotypes. Hierarchical analyses of molecular variance (AMOVA) within and among geographically defined groups tested two a priori hypotheses of structure based on major and minor river drainages, as well as genetic distance clusters defined a posteriori from an unrooted analysis. High genetic differentiation (FST) between populations was found across analyses. The influence of isolation by distance could potentially account for much but not all of the variation found among geographically defined groups at both levels. High variability among the three northernmost genetic clusters (FCT), low variability among populations within clusters (FSC), and low within-cluster Mantel correlations indicate the importance of unidentified likely intra-karst barriers to gene flow separating closely grouped cave populations. Overall phylogeographic patterns are consistent with previous evidence of population isolation among cave systems in the region, revealing geographically structured cryptic diversity in Darlingtonea over its distribution. The landscape features considered a priori in this study were not predictive of the genetic breaks among the three northern clusters, which are genetically distinct despite their close geographic proximity.