Abstract: Cucumber mosaic virus (CMV) is one of most important diseases in tobacco, and selection of molecular markers closely linked to CMV resistant QTLs is a key for tobacco CMV-resistance breeding. In order to identify genes closely linked to CMV resistance and develop resistant varieties, a BC1 population derived from resistant-parent Taiyan-8 and susceptible-parent NC82 was used to construct resistant and susceptible pools. By application of BSA and SLAF-seq technique, a total of 180,370 SALF labels were obtained and 6,156 polymorphic SNP markers were developed. As a result, two CMV-associated regions were identified by analysis of SNP-index data. Our results showed that abundant polymorphic SNPs can be developed between tobacco varieties using SALF-seq. By utilization of these SNPs, two CMV-resistance QTLs were successfully identified in Chr01 and Chr18, with 4.72 Mb and 4.49 Mb interval, respectively. The two identified QTLs are useful for in molecular marker assisted selection in tobacco breeding programs for CMV resistance.