The nematodes Abbreviata antarctica von Linstow, 1899, and Abbreviata hastaspicula Jones, 1979, are predominant spirurid nematodes in species of Varanus lizards in Australia. However, genetic knowledge of these two species of nematode is lacking. In this study, nematodes removed from Varanus gouldii were examined using integrated morphologic and molecular methods. We extracted DNA from A. hastaspicula and A. antarctica for PCR and sequencing. Specific 18S small subunit ribosomal RNA (rRNA) primers were designed on the basis of existing 18S rRNA sequences of Physalopterinae strains. Species of Abbreviata, which are closely similar morphologically, may be misidentified, especially the larvae of different species of Abbreviata that cannot be differentiated. The findings of this study will improve the accuracy in identification of A. hastaspicula and A. antarctica, both morphologically and molecularly.