Stable carbon and hydrogen isotopes of wood methoxy groups (δ2Hmeth, δ13Cmeth), mainly sourced by structural biomolecules like lignin and pectin, provide important insights into climatic, hydrological and physiological conditions. This study systematically investigated species‐specific δ2Hmeth and δ13Cmeth variations in leaves and woody twigs of 65 different tree species grown in a common garden. Significant phylogenetic patterns were observed in δ2Hmeth and δ13Cmeth of both tissues, with stronger signals in leaves and the most pronounced differences between angiosperms and gymnosperms. δ13Cmeth variations are likely explained by anatomical and physiological differences between seed types, while δ2Hmeth variations were attributed to temporal differences in water uptake or isotope fractionation processes. Notably, δ13Cmeth values were more negative in leaves than in twigs, while δ2Hmeth values showed no tissue‐specific difference. This suggests that serine, a methoxy precursor, is differently synthesised in autotrophic than in heterotrophic tissues. Hydrogen isotope fractionation between xylem water and twig methoxy groups averaged at −197 mUr, with mean isotope fractionation of gymnosperms −209 mUr being significantly different to that of angiosperms −184 mUr. Weak relationships between δ2Hmeth and δ2H values of carbohydrates indicated that distinct signals are preserved within the two compounds. This study highlights the importance of phylogenetic considerations when using methoxy group isotopes as proxies and provides new insights into methoxy group biosynthesis.