The root-knot nematode (RKN) Meloidogyne incognita is a major constraint in cotton (Gossypium hirsutum L.) production in numerous countries. Control of RKN has been hampered by the lack of options for crop rotation and even more, by the lack of high-quality locally-adapted varieties exhibiting high levels of resistance. High levels of resistance occur in breeding lines, but the high level of resistance has not readily been transferred to cultivated varieties. Furthermore, cotton genetic improvement is based on the exploitation of a single source of resistance, making the crop vulnerable to the selection of resistant isolates. Resistance to RKN is also found in wild tetraploid cotton (G. hirsutum and G. barbadense) accessions that represent valuable resources for novel genes to be used for cotton improvement. Accessions of Gossypium spp. were evaluated for resistance to RKN in greenhouse experiments, and a highly resistant accession of G. barbadense displaying significant reduction in nematode reproduction was identified. Histological observations of this highly resistant G. barbadense accession (CIR1348) showed that resistance occurs through a two-stage mechanism involving a hypersensitive-like response. Accession CIR1348 showed high levels of resistance to various isolates of RKN originating from the major cotton-producing regions of Brazil. Genetic analyses indicate that resistance is recessive, and controlled by two major genes. The highly resistant accession CIR1348 was crossed with a susceptible cultivated variety (FiberMax966) to produce an F2 mapping population and to initiate the introgression of resistance into an adapted genetic background. The QTL analysis identified 2 major QTLs: one QTL is localized on chromosome 11, previously reported to harbor RKN-resistance loci in other sources of resistance, while the second one resides on a chromosome not previously identified as harboring RKN resistance genes/loci. The identification of SSR markers associated wi