The evolutionary relationships of the different groups of nematodes within the order Strongylida based on morphological data have been speculative and the subject of conjecture. In this paper, we present a multigene phylogenetic analysis, using sequence data of the 18S and 28S ribosomal RNA genes from representatives of all four suborders and seven superfamilies of the Strongylida, to test existing hypotheses proposed for the relationships of the suborders based on morphological data sets. The results obtained demonstrated that the Strongylida is a monophyletic assemblage, with only the Metastrongylina (but not the other suborders) forming a distinct monophyletic clade. We show that, in contrast to all previous hypotheses, one major lineage comprises taxa which occur exclusively in the pulmonary, circulatory or nervous systems of marsupial and eutherian mammals, whereas a second lineage comprises species occurring in the gastrointestinal tracts or perirenal tissues of vertebrates, or in the lungs of birds. The findings suggest that the predilection site of adult nematodes and host type reflect the evolutionary origin of the different taxonomic groups within the Strongylida.