Hydrogen isotopes of plant‐derived biomarkers can vary by >100‰ at a single location. Isotope fractionation associated with the movement of water in plant leaves cannot account for this variability alone. Biochemical processes therefore must play a fundamental role in controlling hydrogen isotope fractionation during secondary compound biosynthesis. Different biosynthetic pathways utilize discrete hydrogen pools and occur within distinct cell compartments. We analyzed hydrogen isotope compositions of C16 and C18 fatty acids and phytol from seven salt marsh plants and compared these data with (i) leaf water and n‐alkane δ2H, (ii) leaf carbon and nitrogen contents, and (iii) nitrogen isotopes of bulk tissue, to evaluate the relationship between biochemical processes, cellular compartmentalization, and hydrogen isotope fractionation. Interspecies variation in chloroplastic fatty acids and phytol δ2H exceeds leaf water δ2H, indicating that different commitments of metabolites among species at branching points in chloroplast metabolic processes may be important determinants of lipid δ2H values. Dominant osmoregulatory strategies, in particular, show strong correlation with leaf wax n‐alkane δ2H. Species that preferentially produce nitrogenous compounds (dicots/shrubs) as protective solutes have 2H‐enriched n‐alkanes relative to species that produce mainly carbohydrates (monocots). n‐Alkane δ2H values, in combination with δ15N data and elemental (C, N) composition, together provide information about biochemical environmental adaptations exhibited by different higher plant species in response to environmental stresses. Thus, while spatial and temporal integration of biomarkers may produce an isotopic record of ecosystem function, biomarkers from individual plant or microbial remains may hold additional details into biologic function and adaptation to ancient environments.