Abstract: With the large-scale cultivation of poplar in China and worldwide, disease management has become an increasingly critical challenge, particularly for anthracnose caused by Colletotrichum gloeosporioides （Cg）, which poses a serious threat to forestry production. Although previous studies have revealed certain aspects of the pathogen’s infection mechanisms, the resistance and susceptibility responses of poplar at the metabolic and transcriptional levels have not been systematically elucidated. In this study, a resistant genotype （Populus × canadensis, Pc） and a susceptible genotype （Populus alba × P. glandulosa, 84K） were used as experimental materials. Pathogen inoculation assays combined with integrated transcriptomic and metabolomic analyses were performed to compare their molecular response profiles before and after pathogen stresses. The results showed that prior to pathogen challenge, Pc exhibited significantly higher expression levels of defense-related genes and greater accumulation of corresponding metabolites in several key pathways （such as phenylpropanoid metabolism and flavonoid biosynthesis） compared with 84K. Following pathogen inoculation, integrated transcriptomic and metabolomic analyses revealed that the defense response in Pc was concentrated in a limited number of critical pathways, with strong consistency observed between changes in gene expression and metabolite accumulation. In contrast, 84K displayed extensive metabolic reprogramming and multi-pathway transcriptional activation, yet showed weaker correlation between gene expression and metabolite profiles. This multi-omics study elucidates the distinct molecular regulatory strategies employed by poplar genotypes with differing resistance levels in response to pathogen stress, providing theoretical insights into the disease resistance mechanisms in poplar.