Widespread mortality of eastern hemlock, Tsuga canadensis (L.) Carrière, resulting from infestation by hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), has occurred throughout the native range of eastern hemlock within the eastern United States. Imidacloprid, a systemic insecticide, is one of the primary chemical compounds used to control hemlock woolly adelgid in both urban and, in a limited manner, in natural forest environments. The metabolism of imidacloprid in eastern hemlock produces 12 metabolites; two of these, imidacloprid 5-hydroxy and imidacloprid olefin, are considered toxicologically important metabolites. However, little is known about the persistence of these metabolites in eastern hemlock in the southern Appalachians. Concentrations ofimidacloprid, olefin, and 5-hydroxy were quantified by using HPLC/MS/MS techniques. Over the 3-yr study, concentrations of imidacloprid and consequent 5-hydroxy and olefin were highest in trees treated with a soil injection in the spring. Imidacloprid and 5-hydroxy concentrations in sap were highest at 12 mo posttreatment and in tissue at 15 mo posttreatment. Imidacloprid was detected through 36 mo posttreatment and 5-hydroxy was detected through 15 mo posttreatment. Olefin concentrations in both sap and tissue were highest at 36 mo posttreatment and were detected in high concentrations through 36 mo posttreatment. Concentrations of imidacloprid were highest in the bottom stratum of the canopy and lowest in the top stratum. Concentrations of olefin and 5-hydroxy were highest in the top stratum and lowest in the bottom stratum.