Groundwater-obligate taxa (stygobionts) exhibit variable degrees of habitat specialization, ranging from micro-habitat specialists to generalists occurring across multiple habitat types. The karstic Edwards Aquifer is the most biodiverse groundwater ecosystem in North America, and among the most thoroughly studied. However, little is known about the distribution of stygobionts in the hyporheic zone of spring-fed rivers downstream from the aquifer. The goals of this research were to 1) assess the potential of the hyporheic zone as habitat for ‘karst-adapted’ stygobionts off-karst, and 2) to determine how stygobiont diversity and abundance are related to spatial (i.e., distance from karst) and environmental effects in the hyporheic zone of a spring-dominated river system. We enumerated stygobionts and quantified environmental characteristics for 55 hyporheic samples from 11 sites in the San Marcos River between three and 140 river kilometers downstream from the rivers’ karstic Edwards Aquifer spring source. Linear mixed effects models and variance partitioning were used to investigate relationships between community metrics (richness, abundance, and diversity) and spatial/ environmental parameters. Eight stygobiontic taxa were recovered in hyporheic samples, but both stygobiont richness and diversity decreased with increasing distance from karst. Stygobiont richness was positively correlated with proximity to karst and hydraulic conductivity, which covaried and explained similar amounts of model variance. Stygobiont abundance was positively correlated with hydraulic conductivity and dissolved oxygen, which also explained similar amounts of model variance. Stygobiont diversity was positively correlated with proximity to karst and dissolved oxygen and negatively correlated with temperature, all of which covaried. Stygobiont communities in the hyporheic zone can be structured by both local environmental conditions, via habitat filtering, and by spatial effects (i.e., proximity to adjacent aquifers). The Edwards Aquifer appears to exert a major influence on community structure in the downstream hyporheic zone directly (as a source population for some stygobionts) and indirectly (influencing environmental conditions like hydraulic conductivity and temperature). Habitat specificity is poorly characterized for most rare stygobiont taxa, inhibiting effective conservation planning and implementation, but studies like this expand our understanding of occupied habitats. Maintaining connectivity between rivers and adjacent groundwater aquifers is critical for groundwater-obligate hyporheic residents.