Potato (Solanum tuberosum) is regarded as one of the single most important vegetable crops in South Africa, with an average annual production of 2 million metric tons. The potato industry contributes to an average of $ 0.37b worth of potatoes annually. Over the years, potato production in South Africa has been affected by, among other factors, diseases and plant parasitic nematodes particularly root-knot nematodes (Meloidogyne spp.). In infected potato fields, root-knot nematodes cause great damage to the crop leading to substantial losses in yield and compromised produce quality. The direct and indirect damage caused by Meloidogyne species results in revenue loss due to a high number of table and processing potatoes rejected in markets both locally and internationally. The presence of resistance breaking Meloidogyne populations, the withdrawal of methyl bromide and lack of commercially grown resistant cultivars suggests that growers are likely to experience more losses in the future. Furthermore, distribution of seed tubers harbouring root-knot nematodes, which may also be asymptomatic, inadvertently facilitates transmission of these parasites to new areas thus perpetuating the problem. Therefore, for the potato industry to adequately address the threat of root-knot nematodes, accurate identification and quantification of root knot nematode levels in the field as well as in seed tubers is of importance. Currently most methods of identifying Meloidogyne species largely rely on the use of morphological traits. However, it can be a challenge to accurately differentiate between closely related species using morphology and other classical methods. To resolve this, recent trends globally have focused on the development of DNA-based diagnostics to rapidly and accurately identify different Meloidogyne species. This study therefore sought to; (a) develop a PCR-based diagnostic tool for accurate detection and identification of various Meloidogyne species parasitizing potatoes in South Africa; (b) use this tool to map their distribution and; (c)develop real-time PCR (qPCR) techniques for accurate quantification and characterization of tropical Meloidogyne species from infected potato tubers. In this study, of the 78 composite potato tuber samples collected from various potato growing regions across seven provinces, 24% were found infected with M. javanica, 23% with M. incognita, 17% with M. arenaria, 14% with M. enterolobii, 3% M. chitwoodi, 1% M. hapla and 1% as M. artiellia. The identity of the remaining 17% could not be established. The three tropical species; M. javanica, M. incognita and M. arenaria were identified as the dominant species, occurring almost in every region sampled. Meloidogyne hapla and M. enterolobii occurred in Mpumalanga and KwaZulu–Natal respectively while M. chitwoodi was isolated from two growers located within the Free State. In the study the use of HRMC and real-time PCR was also developed for identification and quantification of tropical Meloidogyne species infesting potato tubers. Using these two techniques, we were able to show that Meloidogyne arenaria populations produced specific melting peaks (79.3183± 0.0295°C, P < 0.05) thus distinguishing themselves from M. incognita (79.5025± 0.0224°C, P < 0.05) and M. javanica (79.96 ± 0.0459°C, P < 0.05). Real-time PCR was also able to detect 1.53/100th of a nematode using second stage juveniles.