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Atlantic bluefin tuna populations are in steep decline, and an improved understanding of connectivity between individuals from eastern (Mediterranean Sea) and western (Gulf of Mexico) spawning areas is needed to manage remaining fisheries. Chemical signatures in the otoliths of yearlings from regional nurseries were distinct and served as natural tags to assess natal homing and mixing. Adults showed high rates of natal homing to both eastern and western spawning areas. Trans-Atlantic movement (east to west) was significant and size-dependent, with individuals of Mediterranean origin mixing with the western population in the U.S. Atlantic. The largest (oldest) bluefin tuna collected near the northern extent of their range in North American waters were almost exclusively of western origin, indicating that this region represents critical habitat for the western population.