Southern root-knot nematodes, Meloidogyne incognita, feed on the underground portions of hundreds of plant species and affect nutrient partitioning and water uptake of the host plants. Sorghum (Sorghum bicolor) is often not significantly damaged by southern root-knot nematodes (RKN) but some sorghum genotypes support greater population densities of RKN than other genotypes. These higher nematode populations increase the risk of damage to subsequently planted susceptible crops. A previous study identified a major quantitative trait locus (QTL) for RKN resistance on sorghum chromosome (chr.) 3. To maintain long-term resistance, multiple resistance genes should be pyramided in a cultivar. In this study, we identified a new source of RKN resistance, created a mapping population, and identified single-nucleotide polymorphism markers using genotyping-by-sequencing of the segregating population. Use of single-marker analysis and composite interval mapping identified a single QTL on chr. 5 that was associated with egg number and egg number per gram of root from the resistant sweet sorghum line PI 144134. This region on chr. 5 and the prior QTL on chr. 3 can be potentially moved from PI 144134 and Honey Drip, respectively, into elite sorghum germplasm via marker-assisted selection for more durable resistance.