Among the most damaging root-knot nematode species, Meloidogyne exigua is especially common in Latin America and constitutes a major agronomic constraint in all major coffee-growing (Coffea arabica) areas. Growing nematode-resistant coffee represents the most promising option for control of the pest. The present study aimed to determine the mode of inheritance of the M. exigua resistance transferred into C. arabica from a related species, Coffea canephora, and to identify associated molecular markers. Segregation data analysis of F2 progeny derived from a cross between the resistant introgression line T5296 and the susceptible accession Et6 showed that the resistance to M. exigua is controlled by a simply inherited major gene (designated the Mex-1 locus). The gall index distribution exhibited by the F2 individuals suggested incomplete dominant expression. Fourteen AFLP markers were found associated with the resistance to M. exigua and a localized genetic map of the chromosome segment carrying Mex-1 was constructed. Furthermore, the association of the identified AFLP markers with Mex-1 was confirmed by analysis of a set of genotypes involving 28 introgression Arabica lines either resistant or susceptible to M. exigua in field conditions. These results represent an important starting point to enhance backcross breeding programmes and to perform an early selection of resistant seedlings.