Severity and acreage of damage by the reniform nematode (Rotylenchulus reniformis) is a significant and increasing problem for US cotton producers. No Upland cotton (Gossypium hirsutum) cultivar has high resistance to the reniform nematode, so external or engineered sources of resistance are needed. The wild African 13-chromosome species G. longicalyx related to cotton is extremely resistant to this pest and presents a possible solution. Two tri-species hybrids that involve G. longicalyx and G. hirsutum, but differ in the third parent -- either G. armourianum or G. herbaceum, respectively, were developed through intensive wide-cross breeding efforts at the USDA, College Station, and subsequently used as starting points for backcrossing into G. hirsutum. Chromosomal abnormalities were expected to abound in early backcross generations, based on the ADFD and ADAF genomic compositions of these two hybrids, respectively. Chromosome numbers and meiotic metaphase-I configurations that were assessed for most of the BC1F1, BC2F1, BC1S1, BC3F1, BC4F1 and BC5F1 plants revealed progression toward 100% 52-chromosome individuals and increasing frequencies of resistant plants that form 26 bivalents at metaphase-I. Highly resistant plants with normal or nearly normal chromosomal constitution have been selected, yielding high levels of reniform nematode resistance into prospectively elite upland cotton. To identify closely linked molecular marker loci that could be used to screen for the resistance gene and employ marker-assisted selection (MAS), we screened SSR markers across 12 highly resistant and 12 highly susceptible plants from a population of 150 classified BC2F1 plants. Screening of the putatively linked markers across reniform nematode bio-assayed plants from advanced backcross generations have identified linked SSR markers, thus revealing the chromosomal location and map position of the reniform nematode resistance gene in cotton.