Abstract: To understand the diversity and genetic structure of tobacco germplasm resources and provide an important theoretical foundation for resource use, GBS (genotyping-by-sequencing) technology was used to discover SNP loci for 92 tobacco germplasm resources. With the SNPs genotyping data, the genetic diversity and genetic structure were analyzed. By using GBS, a total of 147.165 Gb of sequences was generated from the 92 tobacco germplasms, and each sample produced 1.599 622 Gb in average. After being screened, a total of 93 685 of high-quality SNP sites was retained. The range of number of alleles (Na) for different geographic sources of tobacco population was 1.27-1.93. The observed heterozygosity (Ho) was 0.22~0.76. The expected heterozygosity (He) was 0.32-0.59. The number of polymorphic loci was 15 877-44 249. The percentage of polymorphic loci (PPL) was 26.61%-74.17%. The genetic diversity index (H) was 0.19-0.52. The genetic distance (GD) was -0.0148-0.3849. The genetic distance-based NJ clustering was used to classify the materials into two groups. The results of the population structure based on a model-based method indicated that these materials could be divided into four subgroups. In summary, the sequencing data obtained by GBS technology provide an effective and high feasible approach to assist the genotyping of 92 tobacco accessions with high population polymorphism and low genetic diversity abundance. The division of population structure was not completely related to the geographic origin of the germplasm.