Here we investigated changes in primary metabolism and cell death around oviposition sites in two hybrid clones of Eucalyptus with different degree of resistance to Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae), as well as, tolerance to water deficiency. We showed that apices of the resistant clone with oviposition had a higher content of amino acids organic acids, and the compound putrescine, than those of the susceptible clone with oviposition. In contrast, apices of the resistant clone with oviposition had lower sugar content and pyruvate organic acid content than those of the susceptible clone with oviposition. Small areas of necrosis were induced around the oviposition sites in the stem apices of Eucalyptus 24 h after infestation. The resistant clone developed larger necrotic areas that showed progressive increases 24–72 h after infestation than the susceptible clone, in which cell death was significantly lower and no changes were observed in necrotic area over time. Thus, the programmed death of cells around the egg, modulated by several amino acids, is likely the first defence response of Eucalyptus against L. invasa. Our results serve as the basis for the early identification of key metabolites produced in plants in defence against galling insects.