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Background  Tuna species of the genus  Thunnus , such as the bluefin tunas, are some of the most important and yet most endangered trade fish in the world. Identification of these species in traded forms, however, may be difficult depending on the presentation of the products, which may hamper conservation efforts on trade control. In this paper, we validated a genetic methodology that can fully distinguish between the eight  Thunnus  species from any kind of processed tissue.    Methodology  After testing several genetic markers, a complete discrimination of the eight tuna species was achieved using Forensically Informative Nucleotide Sequencing based primarily on the sequence variability of the hypervariable genetic marker mitochondrial DNA control region (mtDNA CR), followed, in some specific cases, by a second validation by a nuclear marker  rDNA first internal transcribed spacer (ITS1) . This methodology was able to distinguish all tuna species, including those belonging to the subgenus  Neothunnus  that are very closely related, and in consequence can not be differentiated with other genetic markers of lower variability. This methodology also took into consideration the presence of introgression that has been reported in past studies between  T. thynnus ,  T. orientalis  and  T. alalunga . Finally, we applied the methodology to cross-check the species identity of 26 processed tuna samples.    Conclusions  Using the combination of two genetic markers, one mitochondrial and another nuclear, allows a full discrimination between all eight tuna species. Unexpectedly, the genetic marker traditionally used for DNA barcoding,  cytochrome oxidase  1, could not differentiate all species, thus its use as a genetic marker for tuna species identification is questioned.

A Validated Methodology for Genetic Identification of Tuna Species (Genus Thunnus)