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The taxonomy of Australian Isoodon bandicoots has changed continuously over the last 20 years, with recent genetic studies indicating discordance of phylogeographic units with current taxonomic boundaries. Uncertainty over species relationships within southern and western Isoodon, encompassing I. obesulus, I. auratus, and I. fusciventer, has been ongoing and hampered by limited sampling in studies to date. Identification of taxonomic units remains a high priority, as all are threatened to varying extents by ongoing habitat loss and feral predation. To aid diagnosis of conservation units, we increased representative sampling of I. auratus and I. fusciventer from Western Australia (WA) and investigated genetic relationships of these with I. obesulus from South Australia (SA) and Victoria (Vic) using microsatellite markers and mitochondrial DNA. mtDNA analysis identified three major clades concordant with I. obesulus (Vic), I. auratus, and I. fusciventer; however, I. obesulus from SA was polyphyletic to WA taxa, complicating taxonomic inference. Microsatellite data aided identification of evolutionarily significant units consistent with existing taxonomy, with the exception of SA I. obesulus. Further, analyses indicated SA and Vic I. obesulus have low diversity, and these populations may require more conservation efforts than others to reduce further loss of genetic diversity.