Atlantic tarpon, Megalops atlanticus, are large migratory fish that make use of a wide array of habitats during the course of their lifetime, including oceanic, coastal and upper estuaries. Many aspects of the migratory ecology of this species are poorly described, including the scope of individual variability in movements across estuarine gradients. Population abundances have declined precipitously in recent decades leading to its classification as "Vulnerable" under the IUCN, lethal methods of identifying migration patterns such as otolith analyses are not feasible. We therefore examined the non-lethal alternative method of combined stable isotope and trace element analysis in subsamples of scales removed from living fish collected in subtropical areas of the western Gulf of Mexico and Puerto Rico. We found significant differences in chemical signatures from edges of scales collected from separate geographic regions. Within scales, we observed consistent enrichments in δ15N values indicating ontogenetic trophic shifts and individual variation in the remaining proxies (Sr:Ca, Ba:Ca, and δ13C) that suggested differential patterns of movement between marine and fresh waters. Notably, values of δ13C were positively related to Sr:Ca in all geographic regions, suggesting that scale δ13C values primarily reflected movement across salinity gradients rather than fractionation associated with trophic increases. Multi-proxy chemical analysis of Atlantic tarpon scales therefore provides a non-lethal alternative to otolith geochemistry for identifying individual movement and trophic patterns of this highly mobile species. © 2014 Elsevier B.V.