A new root-knot nematode (RKN), Meloidogyne izalcoensis, was detected on coffee in Brazil. This species was first described on coffee from El Salvador and later detected in Asia and Africa. There are no reports on the genetic diversity of this species and resistance to M. izalcoensis has not yet been studied in coffee cultivars. All populations were identified by esterase phenotype and sequence-characterized amplified region (SCAR) species-specific markers. Based on random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers, low intraspecific variability was detected among M. izalcoensis populations from Africa, Vietnam and Brazil, but the population from El Salvador showed fewer genetic differences from the other populations. Phylogenetically, all populations of M. izalcoensis from different locations (El Salvador, Kenya, Tanzania, Vietnam and Brazil) grouped with 90% and 69% bootstrap support for COII and HSP90 regions, respectively, indicating that these markers are highly conserved for the species. In addition, both markers enabled separation of M. izalcoensis populations from other important coffee Meloidogyne species, including M. exigua, M. paranaensis, M. incognita, M. arabicida and M. lopezi. In resistance studies, most genotypes tested that had resistance to other Meloidogyne spp. (Amphillo × Catuaí, Hybrid of Timor, IAPAR 59, IPR 99, IPR 100, IPR 102, IPR 103, IPR 105, IPR 106, IPR 107 and IPR 108) were found to be susceptible to M. izalcoensis, except for the rootstock cv. Apoatã IAC 2258, which proved to be moderately resistant, with a genetic segregation for this character of 43.8%.