Abstract: To elucidate the drought tolerance mechanism of the root system in flue-cured tobacco Heluo 1, this study conducted a comparative analysis using Yunyan 87, K326 and Zhongyan 100 as controls. Under drought stress conditions, seed germination, root development, antioxidant capacity, and root transcriptomics were systematically compared and analyzed. The results showed that under different concentrations of PEG-6000, seed germination and root development in all varieties were inhibited, with the inhibitory effect intensifying as concentration increased. However, Heluo 1 exhibited significantly higher seed germination rate and better root growth performance compared to the control cultivars. Root architecture scanning indicated that its total root length, root surface area, and root volume were significantly greater than those of the controls. Furthermore, Heluo 1 displayed lighter Trypan Blue and nitroblue tetrazolium(NBT) staining intensity at the root tips, coupled with significantly higher activities of antioxidant enzymes. Comparative transcriptomic analysis of the root systems under drought stress between Heluo 1 and Zhongyan 100 shows that 1151 differentially expressed genes(DEGs) unique to Heluo 1 pre-and post-treatment are identified. These DEGs were predominantly enriched in pathways such as lignin degradation, phenylpropanoid and flavonoid biosynthesis. Moreover, among the 2,518 common drought-responsive DEGs shared by both varieties, 259 genes exhibited significant genotypic variation in their expression patterns. Notably, 11 of these genes, including the aquaporin PIP2, which are known to be involved in plant drought resistance pathways, were highly expressed in Heluo 1 and may be closely associated with its enhanced drought tolerance. In conclusion, the root system of Heluo 1 is less affected by drought stress, conferring strong drought tolerance. The biosynthesis of secondary metabolites like phenylpropanoids and the elevated expression of droughtresistant genes such as the aquaporin PIP2 constitute the primary mechanisms underlying its resistance to drought stress.