The freshwater Craterocephalus stercusmuscarum (Atherinidae) has a broad northern and south-eastern Australian distribution, and has been divided into two sub-species. Craterocephalus stercusmuscarum fulvus occurs in eastern and western flowing drainages of the southeast, while C. s. stercusmuscarum occurs in north eastern and northern flowing drainages. Four populations of each sub-species were sampled from different river systems to examine if genetic diversity was consistent with this nomenclature or a previously proposed vicariance hypothesis. Allozyme data did not support the notion that the subspecies were genetically distinct, but the mtDNA data showed that haplotypes from one sub-species, regardless of geography, were reciprocally monophyletic to haplotypes of the other subspecies. Thus, mtDNA genetic diversity was partitioned by prevailing taxonomy and the data suggest that C. s. fulvus populations in eastern and western flowing drainages may have had a relatively recent connection subsequently interrupted by geological events.