Increased knowledge of stock mixing and migration of Atlantic bluefin tuna ( Thunnus thynnus ) is required to properly manage and conserve declining populations. Here, we predicted the nursery origin of giant bluefin tuna (n = 224) present in samples from Canadian waters using stable 13C and 18O isotopes in otoliths. The isotopic composition of milled otolith cores (corresponding to the first year of life) of giant bluefin tuna from three decades (1970s, 1980s, 2000s) and three regions within or adjacent to the Gulf of St. Lawrence was compared with otolith 13C and 18O of yearling bluefin tuna collected from eastern (Mediterranean Sea eastern Atlantic, n = 136) and western (western Atlantic, n = 103) nurseries. Maximum likelihood estimates indicated that greater than 99% of bluefin tuna in our Canadian samples originated from the western nursery. No significant differences in estimates of origin for bluefin tuna were detected among decades or among regions, suggesting little to no mixing of eastern and western populations in the Canadian samples examined. These findings justify the use of catch rates from the Gulf of St. Lawrence area as an index of abundance for the oldest members of the western population.