Natural geochemical markers in the otolith of yellowfin tuna (Thunnus albacares) were used to establish nursery-specific signatures for investigating the origin of fish captured in the western Atlantic Ocean (WAO). Two classes of chemical markers (trace elements, stable isotopes) were used to first establish nursery-specific signatures of age-0 yellowfin tuna from four primary production zones in the Atlantic Ocean: Gulf of Mexico, Caribbean Sea, Cape Verde, and Gulf of Guinea. Next, mixture and individual assignment methods were applied to predict the origin of sub-adult and adult yellowfin tuna from two regions in the WAO (Gulf of Mexico, Mid Atlantic Bight) by relating otolith core signatures (corresponding to age-0 period) to baseline signatures of age-0 fish from each nursery. Significant numbers of migrants from Caribbean Sea and eastern Atlantic Ocean (EAO) production zones (Gulf of Guinea, Cape Verde) were detected in the WAO, suggesting that fisheries in this region were subsidized by outside spawning/nursery areas. Contributions from local production (Gulf of Mexico) were also evident in samples from both WAO fisheries, but highly variable from year to year. High levels of mixing by yellowfin tuna from the different production zones and pronounced interannual trends in nursery-specific contribution rates in the WAO emphasize the complex and dynamic nature of this species’ stock structure and population connectivity. Given that geographic shifts in distribution across national or political boundaries leads to governance and management challenges, this study highlights the need for temporally resolved estimates of nursery origin to refine assessment models and promote the sustainable harvest of this species.