ABSTRACT: Maize (Zea mays L.) is the most essential food crop in the world. However, maize is highly susceptible to drought stress, especially at the seedling stage, and the molecular mechanisms underlying drought tolerance remain elusive. In this study, we conducted comparative transcriptome and physiological analyses of drought-tolerant (CML69) and susceptible (LX9801) inbred lines subjected to drought treatment at the seedling stage for three and five days. The tolerant line had significantly higher relative water content in the leaves, as well as lower electrolyte leakage and malondialdehyde levels, than the susceptible line. Using an RNA-seq-based approach, we identified 10,084 differentially expressed genes (DEGs) with 6906 and 3178 DEGs been annotated and unannotated, respectively. Two critical sets of drought-responsive DEGs, including 4687 genotype-specific and 2219 common drought-responsive genes, were mined out of the annotated DEGs. The tolerant-line DEGs were predominantly associated with the cytoskeleton, cell wall modification, glycolysis/gluconeogenesis, transport, osmotic regulation, drought avoidance, ROS scavengers, defense, and transcriptional factors. For the susceptible line, the DEGs were highly enriched in the photosynthesis, histone, and carbon fixation pathways. The unannotated DEGs were implicated in lncRNAs, including 428 previously reported and 22% putative TE-lncRNAs. There was consensus on both the physiological response and RNA-seq outcomes. Collectively, our findings will provide a comprehensive basis of the molecular networks mediating drought stress tolerance of maize at the seedling stage.