Given the complex nature of freshwater catchment divides and emerging evidence of high levels of genetic diversity, there is great potential for cryptic species to exist among Australian freshwater amphipod groups. Among the chiltoniid amphipods, two congeneric species, Austrochiltonia australis (Sayce, 1901) and A. subtenuis (Sayce, 1902), have been widely recorded across southern Australia yet are poorly known and contentiously defined. A large fragment of the mitochondrial DNA cytochrome c oxidase I (COI) gene was examined and morphological diversity among populations assessed across the reported geographic range of the two putative species. The results confirmed A. australis and A. subtenuis as morphological and molecular species. In addition, two previously undetected and cryptic species from Tasmania are recognised sister species to A. subtenuis and A. australis. Working conclusions provide evidence towards a more comprehensive systematic revision of the Chiltoniidae and present species information relevant to conservation and management efforts of Australian river systems. A key is presented to the chiltoniid amphipods of southern Australia.