A novel procedure for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in commercially canned products has been developed. Three mitochondrial regions were simultaneously amplified by multiplex-Polymerase Chain Reaction, one corresponding to the small rRNA 12S subunit as a positive amplification control and two species-specific fragments corresponding to cytochrome b for A. rochei and ATPase 6 for A. thazard, respectively. Testing of two different detection systems revealed the fluorescence-based approach as the most sensitive. The results demonstrate that this rapid, low-cost methodology is a reliable molecular tool for direct application in the authentication of canned products.