AbstractMeloidogyne enterolobii[the guava root-knot nematode (RKN)] is an emerging plant-parasitic nematode that poses a threat to Upland cotton (Gossypium hirsutum) production in the southeastern United States. Like other RKN spp.,M. enterolobiihas a wide host range and proven ability to overcome resistance sources that have helped protect crops from otherMeloidogynespp., including the southern RKN (Meloidogyne incognita). In this study we evaluated the virulence of two North CarolinaM. enterolobiiisolates on Upland cotton germplasm lines having resistance quantitative trait loci (QTL) to RKN (M240 RNR, MRk-Rn-1) and/or reniform nematode (Rotylenchulus reniformis) (M713 Ren1, MRk-Rn-1) in comparison to their susceptible recurrent parents (DPL61, SG747). Multiple assays using eggs or J2 as inoculum demonstrated that both isolates reproduced equally well on all germplasm lines, producing reproductive factor (RF) values ≥ 6 on the otherwise nematode-resistant lines. Measurements of seedling growth in control and inoculated containers suggested that existing nematode-resistance QTL may offer a level of tolerance toM. enterolobiiinfection that should be further explored in greenhouse and field environments.Meloidogyne enterolobiiinfection of SG747 and MRk-Rn-1 showed nearly identical stages of symptom and nematode development over a time-course of 24 days. These data demonstrate that existing RKN and RN resistance QTL available in elite cotton varieties to producers are most likely insufficient in preventing yield loss due toM. enterolobiiand that future research should focus on (i) understanding theM. enterolobii–cotton interaction at the molecular level, and (ii) screening novel germplasm collections to identify resistance loci.