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Zebra-stem of tomato is a disorder characterized by leaf necrosis, wilting, and a stripped pattern on stems of mature plants. Wilting, necrosis, and death of seedlings are also observed. The physiological and genetic causes of zebra-stem are poorly characterized. Anecdotal evidence has suggested pedigrees with S. pimpinellifolium and bacterial speck resistance in the genetic background are often prone to this disorder. We demonstrate a genetic cause using composite interval mapping and association analysis approaches to define quantitative trait loci (QTLs) that contribute to the disorder. A biparental population of F4 partially inbred families was developed for the initial analysis, and four subsequent backcross or F2 populations were used for subsequent validation. Significant QTLs on chromosomes 5 and 10 were identified, explaining ∼60% and 40% of the variation, respectively. Polymorphisms in the Pto locus are strongly associated with the QTL on chromosome 5. The two loci were derived from different parentage, and a significant interaction effect was demonstrated, resulting in the characteristic zebra-stem symptoms when combined.