The phosphoenolpyruvate carboxylase (PEPC) is a crucial enzyme involved in the primary carbon metabolism of plants, catalyzing the conversion of phosphoenolpyruvate (PEP) to oxaloacetate (OAA), a key intermediate in various biosynthetic pathways. In this study, we identified five Populus trichocarpa PEPCs, renamed PtPEPC1-PtPEPC5, which were classified into two subfamilies, PTPC and BTPC, based on phylogenetic analysis. Our analysis revealed significant segmental duplication events during the expansion of PtPEPCs, providing insights into the evolutionary history of these genes in poplar. The syntenic analysis of PEPCs between poplar and other species, such as Arabidopsis and willow, highlighted both the diversification and conservation of these genes across different plant species. Protein interaction networks, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that PtPEPCs may participate in various metabolic pathways related to carbon flow. Expression analysis under stress conditions, including treatments with abscisic acid (ABA), H