To introgress resistance to Rotylenchulus reniformis from the East African wild diploid species Gossypium longicalyx Hutch & Lee into upland cotton, crosses involving G. hirsutum as the recipient species, G. longicalyx as the donor species and several D genome species (G. thurberi, G. harknessii, G. davidsonii, G. armourianum, G. gossypoïdes, G. aridum, G. raimondii) as bridge species were made. Only the combination [(G. hirsutum x G. thurberi)² x G. longicalyx] gave seeds (10 seeds from 34 crosses) that produced viable plants (9 plants). These hybrids were denoted as HTL. The use of SSR markers revealed the presence in the hybrid of G. hirsutum, G. thurberi and G. longicalyx specific alleles proving the success of the crosses. GISH analysis confirmed this result showing the presence of 13 G. longicalyx chromosomes among the 52 of the hybrid. Three HTL hybrid plants evaluated under controlled conditions following inoculation revealed a high level of resistance to R. reniformis equivalent to that of the diploid donor species. All HTL hybrid plants were self-sterile and photoperiodic. Backcrosses achieved using them as female parents gave seeds that produced viable plants. A total of 13 HTL BC1 plants were tested for their resistance to R. reniformis. Among them, nine were resistant (5 highly resistant and 4 resistant) and four were susceptible. The potential for development of commercial cotton with resistance to reniform nematodes is discussed.