Sequencing of the mitochondrial cytochrome b gene has been used to differentiate three tuna species: Thunnus albacares (yellowfin tuna), Thunnus obesus (bigeye tuna), and Katsuwonus pelamis (skipjack). A PCR amplified 528 bp fragment from 30 frozen samples and a 171 bp fragment from 26 canned samples of the three species were analyzed to determine the intraspecific variation and the positions with diagnostic value. Polymorphic sites between the species that did not present intraspecific variation were given a diagnostic value. The genetic distance between the sequences was calculated, and a phylogenetic tree was constructed, showing that the sequences belonging to the same species clustered together. The bootstrap test of confidence was used to determine the statistical validation of the species assignation, allowing for the first time a quantification of the certainty of the species assignation. The bootstrap values obtained from these results indicate that the sequencing of the cytochrome b fragments allows a correct species assignation with a probability > or =95%.