In order to develop diagnostic tools for plant-parasitic nematodes of sugarcane in South Africa, this work investigated the potential biodiversity of these nematodes in the amount of nucleic acid available, the discrimination of species by a PCR-based method (targeting the ITS1 region) and the effect of some experimental conditions on their detection or discrimination. The ITS1 amplified fragment varied in size, ranging from 400 bp (Meloidogyne javanica) to 1200 bp (Longidorus pisi, Paratrichodorus minor, Xiphinema elongatum, Xiphinema zulu). Some species discrimination was possible by discernible differences in ITS1 amplified fragment sizes. The potential, as well as shortcomings (inhibitions, competition) of this approach are assessed and further steps/outcomes discussed. Part of the work also focused on DNA extraction. Nematode genomic nucleic acid was extracted from five nematode species (Pratylenchus zeae, Helicotylenchus dihystera, Meloidogyne javanica, Xiphinema elongatum and Paratrichodorus lobatus) and we discuss the significant differences obtained between species in the amount of nucleic acid extracted. There were also marked differences in the quantity and quality of nucleic acid extracted from nematode suspensions originating from soils, depending on the extraction buffer used.