Sequence comparisons and molecular phylogenetic analyses were used to describe the nucleotide variability of the ITS containing regions of eighteen Pratylenchus species and several populations. Comparative analysis of nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among Pratylenchus species used in the present study demonstrates that ITS sequences can widely vary in primary sequence and length. Alignment of eighty-seven Pratylenchus sequences and one outgroup taxon reveals the presence of ambiguous regions that have the greatest effect on phylogeny reconstruction. Phylogenetic analyses using Bayesian Inference, Neighbour Joining-LogDet, Maximum Likelihood and Maximum Parsimony, distinguished twelve highly or moderately supported major clades within Pratylenchus. Our results support the taxonomic usefulness of the ITS region to identify root-lesion nematode species of the genus Pratylenchus but the high nucleotide variability, sometimes, can preclude its use to resolve relationships among all members of the genus. In addition, the phylogenetic groupings are not congruent with those defined by characters derived by lip patterns and numbers of lip annuli.