Pearl millet, Pennisetum glaucum, is an important forage crop and a potential feed crop in the southeastern United States. In this region, root knot nematodes, Meloidogyne spp, are significant pests for cotton and peanut. As a possible rotation crop with cotton and peanut in southeastern United States, the knowledge of nematode resistance and its inheritance is important to breed pearl millet for root knot nematode resistance. In this research, quantitative trait loci (QTL) analysis and qualitative trait mapping were conducted to locate resistance to the southern root knot nematode based on a genetic map constructed with AFLP and SSR markers, and 180 recombinant inbred lines (RILs) derived from an intraspecific cross between Tift 99B and Tift 454. One major QTL, Mi-2, having a LOD score of 17.58 and explaining 39.0% of the phenotypic variance , and a nematode resistance gene were located in a 9 cM distal region of pearl millet LG2. Expressed sequence tagged simple sequence repeat (EST-SSR) marker ICMP 3029 (ori), the nearest marker to the nematode resistance gene, could be used in marker-assisted selection (MAS) for nematode resistance.