The Balcones Escarpment of south-central Texas is the eroded topographic expression of an en echelon fault zone where Cretaceous carbonates have been modified by karst processes influenced by structural and stratigraphic controls. While the modern confined Edwards Aquifer flows through cavernous voids at the base of the escarpment, air-filled caves perched in the escarpment are relicts of paleoaquifer hydrology. The structural geology of the Balcones Escarpment and the phylogeography of its endemic cave spiders provide mutually informative frameworks from which to establish relative dates for the activation of discrete groundwater recharge areas. The mitochondrial genetic variability of troglobitic spiders is correlated with the structural elements of the Balcones fault zone in and around the San Antonio relay ramp. Older (basal) genetic lineages occur in structurally high, mature karst terrains, while the younger (derived) lineages occur in structurally low, emergent karst terrains. Based on mitochondrial deoxyribonucleic acid (mtDNA) data, Cicurina diversity is interpreted as the product of the progressive availability of vadose zone habitat as discrete recharge areas have developed.