Abstract: To identify high-quality cigar tobacco germplasm resistant to bacterial wilt (Ralstonia solanacearum) and provide molecular markers for subsequent breeding, this study utilized GBS (genotyping-by-sequencing) resequencing data from 19 tobacco germplasm resources, including the cigar tobacco line ZX145. The number of identified ZX145-specific SNP loci was 221. The specific SNP loci were screened for the development of KASP (kompetitive allele-specific PCR) molecular markers. Validation using 71 tobacco germplasm resources of diverse types yielded 28 KASP marker pairs capable of distinguishing ZX145 from other germplasm. A specific SNP fingerprint for ZX145 was constructed using 24 loci selected to cover all chromosomes. Field evaluations for bacterial wilt resistance, alongside comprehensive assessments of agronomic and yield traits, were conducted in disease nurseries of Fujian, Guizhou, and Anhui Province. The results showed that ZX145 exhibited moderately resistance-resistance in the disease nurseries, which was superior to the conventional control varieties, and its output value performed better. Consequently, ZX145 represents an elite resistant germplasm resource for breeding bacterial wilt-resistant cigar tobacco cultivars. The developed KASP markers provide robust molecular tools to support its utilization in breeding programs.