Zucchini yellow mosaic virus (ZYMV; potyviridae) represents a major pathogen of Cucurbitaceae crops. ZYMV resistance in melon PI 414723 is conditioned by a dominant allele at the Zym locus. This resistant accession restricts viral spread and does not develop mosaic symptoms, but necrosis sometimes develops in response to inoculation. In previous studies, Zym has been mapped to linkage group II of the melon genetic map. In the present study, positional cloning of the locus was undertaken, starting from the CM-AG36 SSR marker at approximately 2 cm distance. We utilized five mapping populations that share the same resistant parent, PI 414723, and analyzed a total of 1630 offspring, to construct a high-resolution genetic map of the Zym locus. Two melon BAC libraries were used for chromosome walking and for developing new markers closer to the resistance gene by BAC-end sequencing. A BAC contig was constructed, and we identified a single BAC clone, from the ZYMV susceptible genotype MR-1, that physically encompasses the resistance gene. A second clone was isolated from another susceptible genotype, WMR 29, and the two clones were fully sequenced and annotated. Additional markers derived from the sequenced region delimited the region to 17.6 kb of a sequence that harbors a NAC-like transcription factor and, depending on the genotype, either two or three R-gene homologs with a CC-NBS-LRR structure. Mapping was confirmed by saturating the map with SNP markers using a single mapping population. The same region was amplified and sequenced also in the ZYMV resistant genotype PI 414723. Because numerous polymorphic sites were noted between genotypes, we could not associate resistance with a specific DNA polymorphism; however, this study enables molecular identification of Zym and paves the way to functional studies of this important locus.