The molecular genetic techniques of allozyme electrophoresis and mitochondrial DNA sequencing were used to examine species boundaries, phylogenetic affinities, and population structure in wolf spiders associated with artesian springs of the Great Artesian Basin (GAB) in South Australia. These springs contain the only permanent water in this extremely arid region, and consequently are of great biological, economic, and sociological significance. Molecular diagnoses of species boundaries in nine lycosid species, involving 56 individuals genotyped at 37 putative allozyme loci and 21 individuals sequenced for a ~600-bp portion of nicotinamide adenine dinucleotide dehydrogenase 1 (NADH1), were largely concordant with those recently proposed on morphological criteria. They also identified a species not previously collected, and suggested that GAB and mesic forms of Venatrix arenaris (Hogg) may not be conspecific. As well as insights into the evolutionary relationships among species and genera, phylogenetic analysis demonstrated two distinct GAB lineages within Venatrix Roewer and Hogna Simon. Population structure analyses of the two most widespread species revealed contrasting patterns. For V. fontis Framenau & Vink, allozyme analyses of 300 individuals at 15 polymorphic loci plus NADH1 sequence analysis of 72 individuals revealed the presence of distinctive subpopulations at most sites, and a partial correlation with overall geographic proximity. In contrast, allozyme analysis of 191 V. arenaris specimens at 12 polymorphic loci demonstrated a comparative lack of both within-site variability and between-site differentiation in the GAB metapopulation.