Diacylglycerol kinase (DGK) is an enzyme that plays a pivotal role in abiotic and biotic stress responses in plants by transforming the diacylglycerol into phosphatidic acid. However, there is no report on the characterization of soybean genes in spite of the availability of the soybean genome sequence. In this study, we performed genome-wide analysis and expression profiling of the gene family in the soybean genome. We identified 12 genes (namely ) which all contained conserved catalytic domains with protein lengths and molecular weights ranging from 436 to 727 amino acids (aa) and 48.62 to 80.93 kDa, respectively. Phylogenetic analyses grouped genes into three clusters-cluster I, cluster II, and cluster III-which had three, four, and five genes, respectively. The qRT-PCR analysis revealed significant gene expression levels in both leaves and roots coping with polyethylene glycol (PEG), salt, alkali, and salt/alkali treatments. This work provides the first characterization of the gene family in soybean and suggests their importance in soybean response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of this gene family.