Tsuga canadensis (eastern hemlock) is an important tree species in North America, but it has been subject to threats from invasive insects such as Adelges tsugae (hemlock woolly adelgid). A. tsugae is native to Asia, and Tsuga species from Asia are typically resistant to this insect. In this study, we examined the phyllosphere microbiome of two hemlock species native to North America and susceptible to A. tsugae ( T. canadensis and T. caroliniana) as well as two species native to Asia and resistant to A. tsugae ( T. chinensis and T. diversifolia). Because pesticide application is often used to treat native Tsuga species impacted by A. tsugae, we also examined the response of the microbiome and overall plant physiology to the pesticide dinotefuran in T. canadensis. We found significant differences in the bacterial and fungal microbiome among the four species of Tsuga. Dinotefuran also significantly affected bacterial communities, but not fungal communities, of T. canadensis. The genus Methylobacterium, which uses methanol produced during plant cell elongation and growth, was a significant indicator of trees not subject to dinotefuran. This suggests that dinotefuran may have reduced cell growth in treated trees. Despite changes in the microbiome, dinotefuran application did not significantly affect plant physiology; however, the coefficient of variation in dinotefuran-treated trees was larger than that in non-treated trees. Our results suggest that the microbiome could play a role in mediating insect resistance in Tsuga species and that pesticide application could affect the phyllosphere microbiome, with implications for plant growth and resistance. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .