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Previously described from only fragments of exoskeleton and juvenile specimens, the cave spider genus <i>Troglodiplura</i> (Araneae: Anamidae), endemic to the Nullarbor Plain, is the only troglomorphic member of the infraorder Mygalomorphae recorded from Australia. We investigated the distribution of <i>Troglodiplura</i> in 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 of <i>Troglodiplura</i> as an independent lineage within the subfamily Anaminae (the ‘<i>Troglodiplura</i> group’) and provide unequivocal evidence that populations from apparently isolated cave systems are conspecifics of <i>T. beirutpakbarai</i> Harvey & 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.