Plants employ a diverse set of defense mechanisms to mediate interactions with insects and fungi. These relationships can leave lasting impacts on host plant genome structure such as rapid expansion of gene families through tandem duplication. These genomic signatures provide important clues about the complexities of plant/biotic stress interactions and evolution. We used a pseudo‐backcross hybrid family to identify quantitative trait loci (QTL) controlling associations between Populus trees and several common Populus diseases and insects. Using whole‐genome sequences from each parent, we identified candidate genes that may mediate these interactions. Candidates were partially validated using mass spectrometry to identify corresponding QTL for defensive compounds. We detected significant QTL for two interacting fungal pathogens and three insects. The QTL intervals contained candidate genes potentially involved in physical and chemical mechanisms of host–plant resistance and susceptibility. In particular, we identified adjoining QTLs for a phenolic glycoside and Phyllocolpa sawfly abundance. There was also significant enrichment of recent tandem duplications in the genomic intervals of the native parent, but not the exotic parent. Tandem gene duplication may be an important mechanism for rapid response to biotic stressors, enabling trees with long juvenile periods to reach maturity despite many coevolving biotic stressors.