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Brown spot (BS) is a destructive foliar disease in tobacco (Nicotiana tabacum L.) and is caused by Alternaria alternata. BS poses a serious threat to tobacco production worldwide. To develop molecular markers that are tightly linked to BS resistance for marker-assisted selection (MAS), F2, F2:3 and BC3F2:3 populations were developed from a cross between a source of BS resistance Jingyehuang (JYH) and a BS susceptible flue-cured variety NC82. One hundred eighty-one F2 individuals, 180 F2:3 lines and 256 BC3F2:3 lines were evaluated for field resistance under different environments and quantitative trait loci (QTL) were identified by linkage mapping. A major QTL was mapped on chromosome15; this QTL explained 8.6-18.0% of the phenotypic variation under different conditions. Furthermore, 219 accessions were evaluated for their responses to BS at two sites, and association mapping (AM) was used to verify the chromosomal region harboring the major QTL. The AM results showed that six significant marker-trait associations were detected at two sites. Among these markers, the marker Indel53 within the specific chromosomal region exhibited the most significant association with resistance to BS and explained 20.0 and 21.5% of the phenotypic variation at the two sites, respectively. An approximately 2-Mb physical interval at the locus of marker Indel53 contained 31 predicted genes; quantitative real-time PCR results suggested that two of these genes (Nitab 4.5_0000264g0050.1 and Nitab 4.5_0000264g0130.1) were probable candidate genes for resistance to BS. In summary, our results suggested that the novel major QTL from tobacco variety JYH for resistance to BS provided partial effective resistance against A. alternata and was useful for MAS of resistance to BS in tobacco breeding. [ABSTRACT FROM AUTHOR]