Although hydrogen isotopes (δ ² H) are commonly used as tracers of animal movement, minimal research has investigated the use of δ ² H as a proxy to quantify resource and habitat use. While carbon and nitrogen are ultimately derived from a single source (food), the proportion of hydrogen in consumer tissues originates from two distinct sources: body water and food. Before hydrogen isotopes can be effectively used as a resource and habitat tracer, we need estimates of (net) discrimination factors (Δ ² H Net ) that account for the physiologically mediated differences in the δ ² H values of animal tissues relative to that of the food and water sources they use to synthesize tissues. Here we estimated Δ ² H Net in captive green turtles ( Chelonia mydas ) by measuring δ ² H values of tissues (epidermis and blood components) and dietary macromolecules collected in two controlled feeding experiments. Tissue δ ² H and Δ ² H Net values varied systematically among tissues, with epidermis having higher δ ² H and Δ ² H Net values than blood components, which mirrors patterns between keratinaceous tissues (feathers, hair) and blood in birds and mammals. Serum/plasma of adult female green turtles had significantly lower δ ² H values compared to that of juveniles, likely due increased lipid mobilization associated with reproduction. This is the first study to quantify Δ ² H Net values in a marine ectotherm, and we anticipate our results will further refine the use of δ ² H analysis to better understand animal resource and habitat use in marine ecosystems, especially coastal areas fueled by a combination of marine (e.g., micro/macroalgae and seagrass) and terrestrial (e.g., mangroves) primary production.