The Yilgarn calcrete aquifers in Western Australia are an interesting system for investigating the process of speciation within subterranean habitats, because of the limited opportunities for dispersal between isolated calcretes. The presence of different-sized diving beetles (Dytiscidae) in separate calcretes, including sympatric sister-species pairs, suggests that species may have evolved within calcretes by an adaptive shift as a result of ecological-niche differentiation. We have studied the potential for trophic niche partitioning in a sister triplet of diving beetles, of distinctly different sizes, from a single aquifer. Fragments of the mitochondrial COI gene, specific to known species of amphipods and copepods, were polymerase chain reaction-amplified from each of the three beetle species, indicating that there is an overlap in their prey items. Significant differences were found in the detected diets of the three species, and results showed a propensity for prey preferences of amphipods by the large beetles and one species of copepod for the small beetles. A terrestrial source of carbon to the calcrete was suggested by stable isotope analyses. The combined approach of molecular, stable isotope and behavioural studies have provided insight into the trophic ecology of this difficult-to-access environment, providing a framework for more fine-scale analyses of the diet of different-sized species to examine speciation underground.