Trembling aspen (Populus tremuloides Michx.) as a clonal tree species possesses a complex root system through which trees of the same or different clones are connected. Root connections have been studied with respect to resource sharing, but the nature, quantities or extent of what is shared between trees is relatively unknown. In this study, we posed the hypothesis that systemic defense induction signals could also spread through these root networks and trigger defenses in neighboring ramets before arrival of pests. Temporal expression pattern of Kunitz trypsin inhibitor (KTI) and dihydroflavonol reductase (DFR) genes, two markers of poplar defense, was followed by quantitative real-time polymerase chain reaction. The expression was quantified in systemic leaves of wounded and healthy plants that shared the same parental root and in untreated controls grown in separate pots. Untreated interconnected plants did not show induced resistance upon herbivore-simulated attack. Although wound-treated ramets induced defense genes, untreated interconnected plants produced an expression pattern similar to non-connected controls. Root connections do not automatically lead to induction of defensive traits that are expressed in plants directly under damage thought to simulate herbivory. Rather, it seems that other communication means such as airborne volatiles can serve as signal transmission pathways among neighboring plants.