Eighty four new and four published ITS1-5.8S-ITS2 sequences of rDNA obtained from different populations of 24 nominal species and 28 isolates of 16 putative Steinernema species were analysed using the maximum parsimony method. In most of the phylogenetic trees obtained from different ITS alignments and phylogenetic procedures, the 84 isolates formed five main, highly or moderately supported, clades, viz Clade I: 'affine-intermedium'; Clade II: 'carpocapsae-scapterisci-tami'; Clade III: 'feltiae-krausseioregonense'; Clade IV: 'bicornutum-ceratophorum-riobrave'; and Clade V: 'arenarium-glaseri-karii-longicaudum'. The ITS rDNA data were found to be of little utility in resolving relationships between these clades, but were useful in studying relationships between species within certain clades. The level of intra-specific variability was different between clades with sequence divergence of 2.4-2.8% of ITS rDNA for some species. Analysis of a combined data matrix of both molecular and morphological features was performed with six qualitative and three quantitative features.