Abstract: In order to discover and locate the genetic locus of resistance to tobacco bacterial wilt, using restriction site-associated DNA sequencing (RAD-seq) technology, and 219 domestic and foreign tobacco varieties with high genetic diversity as the natural population, the resistance to bacterial wilt of the tested materials was identified by field disease nursery, through genome-wide association study (GWAS) to discover disease-resistant genome segments and predict candidate genes for disease resistance. The results showed that 8 tobacco varieties with high resistance to bacterial wilt were identified, and 384 904 high-quality SNP loci were obtained. The distance of genome linkage disequilibrium (LD) attenuation of flue-cured tobacco population was estimated to be 256 kb in average. A segment significantly associated with tobacco bacterial wilt resistance variation was discovered in the tobacco genome, with the peak SNP at 23 977 382 bp on chromosome 17, and the p=6.196×10^-7, which can explain 14.29% of the phenotype variation. Four disease resistance candidate genes were predicted in this segment. This study provides genomic location information for the next step of tobacco bacterial wilt resistance gene cloning and molecular breeding.