Accessions of Gossypium spp., some of them never previously tested, were evaluated for resistance to a local isolate of Meloidogyne incognita race 3 in greenhouse experiments. Nematode infection was characterized based on the galling and egg mass indexes and the reproduction factors (RF). Root-knot nematode reproduction among the newly tested accessions varied from highly susceptible (AS0188, AS0189) to moderately resistant (MT123 no. 3), and some accessions showed highly reduced nematode reproduction (CIR1343, CIR1348, Fai Mui). Histological observations of two resistant accessions (G. barbadense CIR1348 and G. hirsutum TX-25, respectively) showed that resistance occurs through a two-stage mechanism in the first accession and through a single-stage mechanism in the second. Parasitism is blocked early after second-stage juvenile (J2) penetration or during its initial tissue migration (CIR1348) and the development of later-stage juveniles into female adults is suppressed at a later stage (TX-25 and CIR1348). Fluorescence and bright light microscopy showed that root cells surrounding nematodes exhibit a hypersensitivity-like reaction, with the accumulation of presumably phenolic compounds and the presence of necrotic cells that limit the development of nematodes and the formation of giant cells. Underdeveloped giant cells with degenerated cytoplasmic content were found in small numbers in CIR1348 and in large numbers in TX-25, along with deformed nematodes. The full characterization of the defence mechanisms of novel sources of resistance to the root-knot nematode in cotton constitutes a first step towards their use in crop improvement.