The root-knot nematode (RKN) Meloidogyne incognita Kofoid and White 1919, Chitwood 1949 is a major constraint in cotton (Gossypium hirsutum L.) production in numerous countries. Control of RKN has been hampered by the lack of high-quality local varieties exhibiting high levels of resistance as well as the lack of options for crop rotation. High levels of resistance occur in breeding lines, but this high level of resistance has not been readily transferred to cultivated varieties. Resistance to RKN is also found in wild tetraploid cotton accessions that represent valuable resources for novel genes/mechanisms to be used for cotton improvement. In this work, accessions of Gossypium spp. were evaluated for resistance to RKN in greenhouse experiments. Responses to infection by M. incognita varied among the tested accessions, ranging from highly susceptible to resistant. Some accessions displayed a significant reduction in the nematode reproduction. Histological observations of one of the highly resistant G. barbadense accession showed that resistance may occur through two-stage mechanism involving a hypersensitive-like response. The highly resistant accession was crossed with a susceptible one to generate F1 and F2 plants for further genetic studies. Analysis of the response of these F1 and F2 plants to RKN inoculation indicated that resistance is recessive, and controlled by at least one major gene. Analyses using molecular markers associated to known RKN resistance loci showed that the allele(s) involved are different from those previously described. The characterization of the genetics and of the defense mechanisms associated with this novel source of resistance to RKN in cotton constituted a first step towards its use in crop improvement.