Simple SummaryBlind cave spiders of the genusTroglodipluraare large, enigmatic spiders, which show a number of adaptations to an underground existence, including elongated limbs and a complete lack of eyes. They are known only from the Nullarbor Plain, Australia and prior to this study the species were only known from juveniles or fragments of dead spiders. We investigated the distribution ofTroglodiplurain South Australia, providing detailed observations of the behaviour of living adult and juvenile spiders in their natural habitat and in captivity. Given the adaptations of the spiders to an underground existence, plus the barriers to above-ground dispersal posed by the arid climate of the Nullarbor Plain, we expected species to be confined to single caves. However, our molecular evidence showed that the speciesT. beirutpakbaraiwas distributed across multiple caves in South Australia, each separated by between 10–27 km, and that there had been connection between the caves in recent history. These findings provide intriguing evidence for subterranean between-cave dispersal mechanisms for these spiders. A number of threats to cave fauna and the fragile cave ecosystem are posed, and we recommend further research to better define the distribution of vulnerable cave taxa and their conservation profiles.AbstractPreviously described from only fragments of exoskeleton and juvenile specimens, the cave spider genusTroglodiplura(Araneae: Anamidae), endemic to the Nullarbor Plain, is the only troglomorphic member of the infraorder Mygalomorphae recorded from Australia. We investigated the distribution ofTroglodiplurain South Australia, collecting and observing the first (intact) mature specimens, widening the number of caves it has been recorded in, and documenting threats to conservation. Phylogenetic analyses support the placement ofTroglodipluraas an independent lineage within the subfamily Anaminae (the ‘Troglodipluragroup’) and provide unequivocal evidence that populations from apparently isolated cave systems are conspecifics ofT. beirutpakbaraiHarvey & Rix, 2020, with extremely low or negligible inter-population mitochondrial divergences. This is intriguing evidence for recent or contemporary subterranean dispersal of these large, troglomorphic spiders. Observations of adults and juvenile spiders taken in the natural cave environment, and supported by observations in captivity, revealed the use of crevices within caves as shelters, but no evidence of silk use for burrow construction, contrasting with the typical burrowing behaviours seen in other Anamidae. We identify a range of threats posed to the species and to the fragile cave ecosystem, and provide recommendations for further research to better define the distribution of vulnerable taxa within caves and identify actions needed to protect them.