Cucurbit yellow stunting disorder virus (CYSDV) is one of the main limiting factors of melon cultivation worldwide. To date, no commercial melon cultivars resistant to CYSDV are available. The African accession TGR-1551 is resistant to CYSDV. Two major quantitative trait loci (QTLs) have been previously reported, both located near each other in chromosome 5. With the objective of further mapping the gene or genes responsible of the resistance, a recombinant inbred line (RIL) population derived from the cross between TGR-1551 and the susceptible cultivar ‘Bola de Oro’ was evaluated for resistance to CYSDV in five different assays and genotyped in a genotyping by sequencing (GBS) analysis. The major effect of one of the two QTLs located on chromosome 5 was confirmed in the multienvironment RIL assay and additionally verified through the analysis of three segregating BC1S1 populations derived from three resistant RILs. Furthermore, progeny test using the offspring of selected BC3 plants allowed the narrowing of the candidate interval to a 700 kb region. The SNP markers identified in this work will be useful in marker-assisted selection in the context of introgression of CYSDV resistance in elite cultivars.