Tropical tuna fisheries are central to food security and economic development of many regions of the world. Contemporary population assessment and management generally assume these fisheries exploit a single mixed spawning population, within ocean basins. To date population genetics has lacked the required power to conclusively test this assumption. Here we demonstrate heterogeneous population structure among yellowfin tuna sampled at three locations across the Pacific Ocean (western, central and eastern) via analysis of double digest restriction-site associated DNA using Next Generation Sequencing technology. The differences among locations are such that individuals sampled from one of the three regions examined can be assigned with close to 100% accuracy demonstrating the power of this approach for providing practical markers for fishery independent verification of catch provenance in a way not achieved by previous techniques. Given these results, an extended pan-tropical survey of yellowfin tuna using this approach will not only help combat the largest threat to sustainable fisheries (i.e. illegal, unreported and unregulated fishing) but will also provide a basis to transform current monitoring, assessment and management approaches for this globally significant species.