Abstract: The effects of drought stress on root morphology, endogenous hormones, chlorophyll fluorescence and active oxygen metabolism in three different stages of sweet potato rooting, branching and tubering stage were studied by sand culture method with 10% PEG-6000 simulating drought stress. The results showed that the biomass of sweet potato decreased significantly under drought stress in different periods, with the degree of drought stress being 10 days > 20 days > 30 days after transplantation. Drought stress significantly reduced the average diameter and root volume at the 10 days after transplantation, followed by the 20 days and 30 days. Both the main and interactive effects of different periods and drought stress significantly affected root morphological characteristics. Drought stress at different stages significantly reduced chlorophyll fluorescence characteristics, resulting in blocked photosynthate formation, and inhibited root differentiation. Drought stress affected the proportion of endogenous hormones in root (with decreases of indoleacetic acid and zeatin riboside contents but increases of abscisic acid contents) and thus inhibiting root differentiation of sweet potato. The earlier the stress time, the more serious the root differentiation of sweet potato was hindered. Across different drought-tolerant varieties, the severity of root differentiation blocked in Jishu 26 was significantly lower than that in Guangshu 87. Root endogenous hormones and chlorophyll fluorescence were the key drivers for the average diameter and root volume (R₁=0.936, R₂=0.972). Zeatin riboside, maximal photochemical efficiency, and abscisic acid had greater direct effects on average diameter and root volume of sweet potato. Sweet potato was more sensitive to drought stress in the 10th day after transplanting. Therefore, proper irrigation should be considered in case of drought in seedling stage of sweet potato.