Rotylenchulus reniformis is an important root pathogen of cotton in the south‐eastern United States, and management is hindered by the lack of host‐plant resistance in upland cotton (Gossypium hirsutum). The G. arboreum accession PI 417895 is highly resistant to R. reniformis, and a segregating population of 300 F2 plants was developed for phenotypic characterization of resistance. The population showed quantitative variation for nematode infection. Twenty plants showed no infection and were classified as escapes. Fifty‐four plants were classified as resistant or moderately resistant, whereas, 226 were classified as moderately susceptible or susceptible based on the nematode response of the susceptible parent, indicating resistance is a recessive trait, but these data did not support the single recessive gene model. Alternatively, this model would be supported if the 77 plants with a similar nematode response as observed for PI 417895 were classified as resistant. Twelve plants showed high levels of resistance and these data would support a two recessive gene model. Accession PI 417895 represents a new source of R. reniformis resistance with two major genes conferring resistance. Introgression of multiple resistance genes into G. hirsutum will require the development of larger populations to recover the resistant phenotype.