A molecular approach was used to genetically characterize 5 species (Aoruroides queenslandensis, Blattophila sphaerolaima, Cordonicola gibsoni, Desmicola ornata and Leidynemella fusiformis) belonging to the superfamily Thelastomatoidea (Nematoda: Oxyurida), a group of pinworms that parasitizes terrestrial arthropods. The D3 domain of the large subunit of nuclear ribosomal RNA (LSU) was sequenced for individual specimens, and the analysis of the sequence data allowed the genetic relationships of the 5 species to be studiedNucleotide sequence data reported in this paper are available in the GenBank®, EMBL and DDBJ databases under the Accession numbers: AM232755–AM232763.. The sequence variation in the D3 domain within individual species (0–1·8%) was significantly less than the differences among species (4·3–12·4%). Phylogenetic analyses, using maximum parsimony, maximum likelihood, and neighbour-joining, tree-building methods, established relationships among the 5 species of Thelastomatoidea and Oxyuris equi (a species of the order Oxyurida). The molecular approach employed provides the prospect for developing DNA tools for the specific identification of the Thelastomatoidea, irrespective of developmental stage and sex, as a basis for systematic, ecological and/or population genetic investigations of members within this superfamily.