Breeding for resistance to root-knot nematode Meloidogyne exigua in coffee may help in controlling this nematode, which causes substantial harvest losses throughout Latin America. Accessions of Coffea arabica, C. canephora and lines derived from the Timor Hybrid (wild C. arabica × C. canephora interspecific hybrid) were tested in a greenhouse for resistance to gall formation by counting individual galls on the roots of plants inoculated with second-stage juveniles of a M. exigua population from Costa Rica. The level of introgression from C. canephora was also assessed on 28 resistant and susceptible genotypes in a molecular study with amplified fragment length polymorphism (AFLP) markers. The frequency of resistant plants was very low or even nonexistent in C. arabica and very high in C. canephora with > 78% immune plants and 100% resistant plants. Several lines derived from the interspecific hybrid revealed a high level of resistance similar to that observed in the parent C. canephora species. A molecular study of Timor Hybrid-derived lines revealed high variability for the number of markers present in the study genotypes. A larger number of introgression markers was found in the group of resistant genotypes than in the susceptible genotypes. However, there were also lines with little introgression that had retained resistance. Based on genetic distances calculated from these markers, two groups of lines were revealed: those derived from Timor Hybrid CIFC1343, and those derived from Timor Hybrids CIFC832/1 and CIFC832/2, which may indicate the existence of different resistance genes in the two groups. The segregations observed in F1 and F2 progeny may be explained by at least a dominant gene. The lines derived from the Timor Hybrid are a worthwhile source of resistance to M. exigua that can be exploited to improve C. arabica with the help of molecular-assisted selection.