Bottle Gourd, native to Africa, is used as an important rootstock to improve cold tolerance and disease resistance in cucurbits. Viral diseases are considered a major threat to cucurbit crop production. The most prevalent viruses infecting bottle gourd are aphid-transmitted papaya ringspot virus watermelon strain (PRSV-W) and zucchini yellow mosaic virus (ZYMV). The genetic source of resistance to PRSV-W and ZYMV has been identified, but incorporating the disease-resistant gene(s) into a commercial cultivar through traditional breeding would take years or decades. With the recent advancement in the sequencing technologies, we aim to accelerate the molecular breeding for trait improvement. We recently assembled the bottle gourd genome and used genotyping-by-sequencing (GBS) to map the PRSV-W resistance locus in an F2 population. A dominant monogenic locus Prs was mapped in a 317.8-Kb region on chromosome 1 still containing 39 annotated genes. In the current study, through QTL-Seq analysis on pooled libraries with 10 most PRSV resistant or susceptible individuals from six F3 populations, we aim to map the Prs locus and to identify the candidate resistance gene for PRSV-W. In addition, through screening the USDA collections of bottle gourd germplasm, significant variations in the level of resistance to PRSV-W were identified in 154 Plant Introductions (PI) evaluated. Through genome re-sequencing of these PI lines, we intend to use genome-wide association studies to confirm genes or SNPs associated with the PRSV-W resistance in bottle gourd.