Abstract: Flue-cured tobacco variety Zhongchuan208 is a male sterile first-generation hybrid, which is mainly cultivated in central China and Southwest China. In order to enhance the protection and utilization of tobacco varieties, SNP identification was conducted using resequencing data of Zhongchuan208 and its parents, and SNP loci were screened using resequencing data of 5161 germplasm resources as genetic background, and the specific fingerprint of Zhongchuan208 was constructed. The results showed that the fingerprint was composed of 285 SNP loci. After reliability test and actual sequencing verification, the fingerprint had a high tolerance for sequencing errors and locus missing. Under the condition of both reliability and cost, the test samples could be sequenced and tested by 2X or 3X WGS sequencing. In conclusion, the specific fingerprint of flue-cured tobacco variety Zhongchuan208 was successfully constructed in this study. The fingerprint has high reliability and strong practicability, which provides technical support for the promotion and protection of Zhongchuan208, and also provides method reference for the construction of specific fingerprint of tobacco hybrid.