The genus Ditylenchus has been divided into 2 groups: the Ditylenchus triformis-group, and the Ditylenchus dipsaci-group based on morphological and biological characters. A total of 18 populations belong to 5 species of Ditylenchus was studied: Ditylenchus africanus, Ditylenchus destructor, Ditylenchus myceliophagus and dipsaci, Ditylenchus weischeri, the first 3 belong to the Ditylenchus triformis-group, the last 2 the Ditylenchus dipsaci-group. The species of Ditylenchus triformis-group were cultured on fungi, while the species from Ditylenchus dispaci-group cultured on excised roots of plant hosts in petri dish. DNA sequences of regions of the nuclear ribosomal first internal transcribed spacer (ITS1) and the small subunit 18S were PCR amplified, sequenced and the phylogenetic analyses also including the sequences of the closely related species from the GenBank. The randomly amplified polymorphisms of genomic DNA (RAPD) were also generated. Two clusters or clades corresponding to the 2 groups were consistently observed with significant statistical support from the 3 datasets. The phylogenetic analysis also revealed that the genus is paraphyletic, separating the 2 groups by species of Anguina and Subanguina.