Three species of Globocephaloides, parasitic nematodes occurring in macropodid marsupials in different areas of Australia, were characterized by the sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. Samples were subjected to PCR-coupled SSCP analysis and targeted sequencing, in order to assess genetic variation within and among individuals from different host species. Both SSCP and sequence data supported the current classification of morphospecies. Contrary to a previous hypothesis that cryptic species exist within the Globocephaloides trifidospicularis "complex", no or minor (0.2%) variation was detected among individuals from different hosts or geographical origins. Within G. macropodis populations, there was a consistent difference in both the ITS-1 (5.2%) and ITS-2 (7.1%) sequences between individuals derived from Macropus agilis and those from Macropus dorsalis. Although the results suggested that G. macropodis from each host species represented sibling species, future morphological study of individuals representing each G. macropodis genotype is warranted to provide further support for this hypothesis. (Nucleotide sequence data have been deposited in the GenBank database under accession nos. GQ131400-GQ131409.).