The Yilgarn Region of Western Australia contains a rich diversity of subterranean invertebrates and comprises hundreds of physically isolated calcrete aquifers, resembling a subterranean archipelago. Each calcrete has a unique combination of aquatic species including diving beetles (Dytiscidae). Two different genera of diving beetle are found in the aquifers: Paroster and Limbodessus within which ~100 species have now been described. Phylogenetic analyses based on mitochondrial DNA (mtDNA) suggest that most (~85%) of the species have independently evolved from surface ancestors, but also provide evidence for the existence of 11 sister species pairs and two sister species triplets living in sympatry within the same calcrete. This finding raises the possibility that speciation occurred underground from a common ancestor already pre-adapted to subterranean life. Here we focused on Limbodessus and aimed to test the sister species status of key taxa using multiple independent nuclear DNA markers. A further aim was to provide a robust phylogeny for the genus using seven genes, three mitochondrial (COI, 12S, 16S) and four nuclear (CN, WG, TOPO, ARK). These genes were sequenced for 55 species of Limbodessus, three species of Paroster and one species of Allodessus; the latter two genera were used as outgroups. Phylogenetic analyses of individual genes for the 55 Limbodessus species included 13 that had previously been suggested to be part of a sister pair or triplet. These analyses supported the sister species status of five pairs of taxa, but not the status of the triplet, suggesting that potentially there has been some hybridisation in the past leading to introgression of mtDNA between these three species. Overall, our study shows that Limbodessus contains multiple examples of independent speciation underground in either sympatry or parapatry. Further research is required to determine the selective forces that are operating to drive these patterns of speciation.