The host status of South African adapted, genetically modified (GM) glyphosate-tolerant soybean genotypes to root-knot (field and glasshouse) and lesion (field) nematodes were assessed. Analyses of root and soil samples of 29 genotypes (collected from seven production areas during the 2014/15 season) enabled the identification of nine plant-parasitic nematode genera and 10 species. Predominant endoparasitic genera in root samples were Meloidogyne (Meloidogyne incognita and M. javanica) and Pratylenchus (Pratylenchus brachyurus, P. zeae and P. teres). Rotylenchulus parvus was the predominant semi-endoparasite in soil, followed by Scutellonema brachyurus and Helicotylenchus sp. Only ‘PAN 1583 R’ and ‘PAN 1521 R’ maintained less than 10% of the Meloidogyne spp. densities present in roots of the most susceptible genotype, while all genotypes were susceptible to the Pratylenchus spp. The host status of 36 soybean genotypes to M. incognita infection, evaluated in two follow-up glasshouse experiments terminated 56 days after inoculation of ca. 1000 M. incognita eggs and second-stage juveniles (J2) per seedling, varied substantially for final population density (Pf), reproduction factor (Rf) and relative percentage susceptibility (%S). Only ‘PRF-GCI7’ and the resistant reference ‘LS 5995’ had Rfs < 1 for both experiments, despite higher minimum and maximum temperatures recorded for the second experiment. Continuous evaluation of soybean genotypes for their host status to predominant nematode pests and their use to reduce densities of such species in producer’s fields are crucial to enable sustainable crop production, and contribute towards food provision and security.