Methyl salicylate is an important inter- and intra-plant signaling molecule, but is deemed undesirable by humans when it accumulates to high levels in ripe fruits. Balancing the tradeoff between consumer satisfaction and overall plant health is challenging as the mechanisms regulating volatile levels have not yet been fully elucidated. In this study, we investigated the accumulation of methyl salicylate in ripe fruits of tomatoes that belong to the red-fruited clade. We determine the genetic diversity and the interaction of four known loci controlling methyl salicylate levels in ripe fruits. In addition toNon-Smoky Glucosyl Transferase 1(NSGT1), we uncovered extensive genome structural variation (SV) at theMethylesterase(MES)locus. This locus contains four tandemly duplicatedMethylesterasegenes and genome sequence investigations at the locus identified nine distinct haplotypes. Based on gene expression and results from biparental crosses, functional and non-functional haplotypes forMESwere identified. The combination of the non-functionalMEShaplotype 2 and the non-functionalNSGT1haplotype IV or V in a GWAS panel showed high methyl salicylate levels in ripe fruits, particularly in accessions from Ecuador, demonstrating a strong interaction between these two loci and suggesting an ecological advantage. The genetic variation at the other two known loci,Salicylic Acid Methyl Transferase 1(SAMT1) and tomatoUDP Glycosyl Transferase 5(SlUGT5), did not explain volatile variation in the red-fruited tomato germplasm, suggesting a minor role in methyl salicylate production in red-fruited tomato. Lastly, we found that most heirloom and modern tomato accessions carried a functionalMESand a non-functionalNSGT1haplotype, ensuring acceptable levels of methyl salicylate in fruits. Yet, future selection of the functionalNSGT1allele could potentially improve flavor in the modern germplasm.