Diacylglycerol kinase (DGK) is an enzyme that converts diacylglycerol to phosphatidic acid and plays pivotal roles in plant biotic and abiotic stress responses. Although the Populus trichocarpa genome has been sequenced, characterization of the PtDGK gene family has not been reported. In this study, we performed genome-wide investigation and expression pattern analysis of the PtDGK gene family in poplar. In total, 7 PtDGK genes (PtDGK1-PtDGK7) were identified and divided into three clusters, namely, Cluster I (3), Cluster II (2), and Cluster III (2). Sequence analysis revealed that all members contained two conserved domains, DAGKa and DAGKc, while Cluster I contained two additional DAG/PE-binding domains and was more conserved in the protein three-dimensional structure. In addition, the members of the three PtDGK subfamilies had similar gene structures, motif compositions and conserved domains. Chromosome localization revealed that the 7 PtDGK members were located on 7 different chromosomes, with each chromosome containing only one PtDGK member. Two segmental duplication gene pairs were found in Populus trichocarpa, which have undergone purification selection during evolution. Collinearity analysis demonstrated that Populus trichocarpa had more collinear gene pairs with Arabidopsis thaliana than with Vitis vinifera. The promoter regions were rich in hormone and stress response cis-acting elements. RNA-seq transcriptome data revealed different expression patterns of the PtDGKs in different tissues and under various hormone treatments (salicylic acid and jasmonic acid), abiotic stresses (salt, drought, cold and heat), and biotic stresses (pests and pathogens). Quantitative real-time PCR analysis showed that DGK3 and DGK5 can be induced by salt treatment. This work provides the first characterization of PtDGKs in poplar and suggests their importance in biotic and abiotic stresses, which will provide guidance for further research on the understanding and functional dissection of PtDGKs in the future.