Information about the non-target effects of glyphosate, a broad-spectrum systemic herbicide on soil-borne nematodes is scarce and not well documented for South African agricultural fields. In the present study, the abundance and identity of plant-parasitic nematodes in roots and rhizosphere soil of commercial glyphosate-tolerant (genetically modified; GM) and conventional (non-glyphosate-tolerant) soybean cultivars from cultivated fields were obtained for two consecutive growing seasons. Grass and soil from adjacent natural vegetation were sampled, representing the reference system. Thirty plant-parasitic nematode species, belonging to 13 genera were identified in the roots and rhizosphere soil of soybean cultivars and natural vegetation. In rhizosphere soil, Meloidogyne (for glyphosate-tolerant and conventional soybean), followed by Helicotylenchus (for glyphosate-tolerant soybean) and Scutellonema (for conventional soybean), were the predominant genera. Seven species, namely Pratylenchus flakkensis, Pratylenchus scribneri, Pratylenchus vulnus, Rotylenchus brevicaudatus, Telotylenchus avaricus, Tylenchorhynchus brevicaudatus and Quinisulcius capitatus are first reports for soybean in South Africa. Although it was not possible to define the impact of each ecosystem on the plant-parasitic nematode assemblages, this study suggested that glyphosate had no deleterious effects on plant-parasitic nematodes.