Members of the stygofauna are highly specialised invertebrates living within groundwater systems. The hydrographical isolation of groundwater habitats combined with low dispersal abilities of stygofauna has resulted in narrow distributions and high levels of regional endemism. Consequently, stygofauna are particularly vulnerable to disturbance and habitat destruction. Aquifers are under enormous pressure from excessive water extraction, pollution and mining, and the efficient conservation of groundwater ecosystems requires detailed knowledge of dispersal and colonisation capabilities of stygofauna. We investigated fine-scale genetic structure and gene flow in the ancient crustacean Parabathynellidae (Bathynellacea, Syncarida) across a section of the Macquarie River alluvium, New South Wales, Australia. We sampled 175 parabathynellids from eight groundwater monitoring bores at pairwise distances up to 300 m and genotyped each individual at eight microsatellite markers. We found significant intraspecific genetic structuring over short geographical distances (<50 m), supporting the view of limited dispersal in stygofauna. Our study is the first extensive microsatellite study of a stygobiont family, and we have gained an insight into the dispersal patterns of these elusive organisms. Genetic data imply that they rarely colonise new habitats, a trait that renders them susceptible to anthropogenic impacts.