We identified QTLs associated with gummy stem blight resistance in an interspecific F2:3 Citrullus population and developed marker assays for selection of the loci in watermelon. Gummy stem blight (GSB), caused by three Stagonosporopsis spp., is a devastating fungal disease of watermelon (Citrullus lanatus) and other cucurbits that can lead to severe yield losses. Currently, no commercial cultivars with genetic resistance to GSB in the field have been reported. Utilizing GSB-resistant cultivars would reduce yield losses, decrease the high cost of disease control, and diminish hazards resulting from frequent fungicide application. The objective of this study was to identify quantitative trait loci (QTLs) associated with GSB resistance in an F2:3 interspecific Citrullus mapping population (N = 178), derived from a cross between Crimson Sweet (C. lanatus) and GSB-resistant PI 482276 (C. amarus). The population was phenotyped by inoculating seedlings with Stagonosporopsis citrulli 12178A in the greenhouse in two separate experiments, each with three replications. We identified three QTLs (ClGSB3.1, ClGSB5.1 and ClGSB7.1) associated with GSB resistance, explaining between 6.4 and 21.1% of the phenotypic variation. The genes underlying ClGSB5.1 includes an NBS-LRR gene (ClCG05G019540) previously identified as a candidate gene for GSB resistance in watermelon. Locus ClGSB7.1 accounted for the highest phenotypic variation and harbors twenty-two candidate genes associated with disease resistance. Among them is ClCG07G013230, encoding an Avr9/Cf-9 rapidly elicited disease resistance protein, which contains a non-synonymous point mutation in the DUF761 domain that was significantly associated with GSB resistance. High throughput markers were developed for selection of ClGSB5.1 and ClGSB7.1. Our findings will facilitate the use of molecular markers for efficient introgression of the resistance loci and development of GSB-resistant watermelon cultivars.