The accurate identification of root-knot nematode (RKN) species (Meloidogyne spp.) is essential for implementing management strategies. Methods based on the morphology of adults, isozymes phenotypes and DNA analysis can be used for the diagnosis of RKN. Traditionally, RKN species are identified by the analysis of the perineal patterns and esterase phenotypes. For both procedures, mature females are required. Over the last few decades, accurate and rapid molecular techniques have been validated for RKN diagnosis, including eggs, juveniles and adults as DNA sources. Here, we emphasized the methods used for diagnosis of RKN, including emerging molecular techniques, focusing on the major species reported in Brazil.