Drought stress is the major environment constraint on soybean yield, and a variety of pathways underlie drought tolerance mechanisms. Transcriptomic profiling of two soybean cultivars, drought-tolerant SS2-2 and drought-sensitive Taekwang, was performed under normal and drought conditions to identify genes involved in drought tolerance. This revealed large differences in water loss during drought treatment. Genes involved in signaling, lipid metabolism, phosphorylation, and gene regulation were overrepresented among genes that were differentially expressed between cultivars and between treatments in each cultivar. The analysis revealed transcription factors from six families, including WRKYs and NACs, showed significant SS2-2-specific upregulation. Genes involved in stress defense pathways, including MAPK signaling, Ca2+signaling, ROS scavenging, and NBS-LRR, were also identified. Expression of non-specific phospholipases, phospholipase D, andPHOSPHATIDYL INOSITOL MONOPHOSPHATE 5 KINASE(PIP5K), which act in the lipid-signaling pathway, was greatly increased in SS2-2. The roles ofPIP5Kin drought stress tolerance were confirmed inArabidopsis thaliana.Arabidopsispip5kmutants had significantly lower survival rates under drought stress than wild-type plants. This study identified additional elements in the mechanisms used by plants to protect themselves from drought stress and provides valuable information for the development of drought-tolerant soybean cultivars.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11032-023-01385-1.