The aim of the present study was to conduct, in southern Australian waters, a preliminary epidemiological survey of five commercially significant species of fish (yellow-eye mullet, tiger flathead, sand flathead, pilchard and king fish) for infections with anisakid nematodes larvae using a combined morphological-molecular approach. With the exception of king fish, which was farmed and fed commercial pellets, all other species were infected with at least one species of anisakid nematode, with each individual tiger flathead examined being infected. Five morphotypes, including Anisakis, Contracaecum type I and II and Hysterothylacium type IV and VIII, were defined genetically using mutation scanning and targeted sequencing of the second internal transcribed spacer of nuclear ribosomal DNA. The findings of the present study provide a basis for future investigations of the genetic composition of anisakid populations in a wide range of fish hosts in Australia and for assessing their public health significance.