The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is an exotic insect species dramatically reducing populations of eastern hemlock, Tsuga canadensis (L.) Carrieré, throughout the eastern United States. Systemic imidacloprid is one of the primary chemicals used to successfully control infestations of the hemlock woolly adelgid. The concentration levels for this systemic insecticide present in the sap of eastern hemlocks were evaluated from three strata within the canopy over a two year time span. Enzyme-linked immunosorbent assays were conducted every three months posttreatment to assess imidacloprid concentration within the sap. The effect of application timing (fall versus spring) and application method (soil drench, soil injection, and tree injection) on the translocation of imidacloprid throughout the canopy, and the quantity of imidacloprid translocated in the sap of eastern hemlock branches and the terminal twig and needle tissue was determined. Concentration levels progressively declined from the bottom strata to the top strata of the canopy. This trend was consistent in all chemically treated trees. Tree injections provided the lowest concentration and the most nonuniform distribution of imidacloprid throughout the canopy. The highest insecticide concentrations within the tree across all strata over the two year period were consistently associated with the soil drench method followed by the soil injection method. Imidacloprid concentrations peaked between month 9 and 12 posttreatment, and then declined; however, at two years posttreatment, soil drench and soil injected trees contained concentrations reported as being effective for control of the hemlock woolly adelgid.