The relationships of Mustelus species in Australian and New Zealand waters were investigated by analysis of allozymes (28 loci), polymorphisms in the length of restriction fragments from mitochondrial DNA (mtDNA; whole genome, 10 restriction enzymes), and precaudal vertebrae counts. We confirm the existence of four Mustelus species in this region: a nonspotted Mustelus sp. A (a single specimen genetically examined from the North-West Shelf off Western Australia), and three spotted species—M. sp. B (from Western Australia, the North-West Shelf south to Perth—extending its known range); Mustelus antarcticus (from Bunbury in Western Australia around the southern Australian coast and as far north as Townsville, Queensland), and M. lenticulatus from New Zealand. Mustelus sp. A was the most divergent of these species, with fixed differences at six allozyme loci and a unique mtDNA profile in digests with six of the 10 restriction enzymes. The other species were much more similar to each other, with no completely fixed differences detected at any of the allozyme loci assayed. Mustelus sp. B differed very significantly from M. antarcticus in allele frequencies for three allozyme loci, and one restriction enzyme gave a unique mtDNA profile. Mustelus lenticulatus was distinguished from M. antarcticus by significant frequency differences at nine allozyme loci, and two restriction enzymes were nearly diagnostic. MtDNA nucleotide diversity (average 0.0323) and nucleotide divergence (average 0.0315) between species was low. Allozyme variation in M. antarcticus was high (H = 0.100) but low for the other three species (M. sp. A = 0.000; M. sp. B = 0.025; M. lenticulatus = 0.019). Mustelus antarcticus also had the highest mtDNA haplotype diversity of 0.534 (± 0.022); M. sp. B had 0.385 (± 0.149), and M. lenticulatus had 0.000 (this parameter could not be estimated for M. sp. A because only a single specimen was genetically examined). The within-species nucleotide diversities showed a higher value for M. sp. B (0.0021) than for M. antarcticus (0.0014). Significant differences between species were also observed for precaudal vertebrae counts. These are lowest in M. sp. B (77.39 ± 2.79), followed by M. antarcticus (85.61 ± 4.03), M. sp. A (90.50 ± 1.05), and M. lenticulatus (94.88 ± 1.46).