Drought is a major abiotic stress that restricts plant development, especially at seedling stage, which stimulates reduction of shoot, root absorb ability, and biomass accumulation of plant. Peanut, as a plant with resilience to drought stress, can still cause serious condensed shoot and impaired flower bud differentiation under drought conditions resulting in yield loss. In order to find those drought responses of peanut at seedling stage, two distinct cultivars with different drought resistance (HY22 and FH18) were screened previously and used to investigate the difference of drought response by deciphering the changes of morphology, physiology-anatomy, and transcriptome. The results showed that drought stress decreased root morphological indices both in two cultivars, but HY22 showed higher root vigor and lateral roots. Furthermore, HY22 enhanced the activity of antioxidant enzymes such as POD, SOD to reduce lipid peroxidation and H2O2 concentration, in which stable root anatomical structure and microstructure were kept. By contrast, SOD and POD decreased, and MDA and H2O2 increased, in which gave impair anatomical structure and microstructure in FH18. Using high-throughput RNA sequencing, we developed a spatiotemporal transcriptome atlas of HY22 and FH18 at seedling stage. A total of 34,303 and 34,689 genes for HY22 and FH18 were found to be expressed under the drought stress, respectively. Global comparisons of gene expression showed more differentially expressed genes (DEGs) were detected in HY22. Moreover, co-expression analysis of overlapped DEGs between HY22 and FH18 revealed that the up-regulated genes of HY22 mainly related to hormone-mediated signaling pathway, response to abscisic acid and flavonoid biosynthetic process. According to the findings, the genotype HY22 was more tolerant to drought stress than FH18 by improving SOD and POD enzyme activities, membrane stability, root vigor, and maintaining fine cell microstructure and relatively up-regulated express levels of genes and metabolin revolving in ABA transduction signal, terpenoids skeleton and flavonoid biosynthesis. The results of the current study provide theoretical reference for elaborating drought resistance mechanism more accurately and precisely.