Four Donax species, D. semistriatus, D. trunculus, D. variegatus and D. vittatus, are found on European coasts. Nevertheless, despite their economic importance there is not a reliable method to differentiate these species independently of their size or condition. Such a method could help to protect consumer rights and avoid commercial fraud due to the replacement of valuable species by less valuable ones with similar morphological traits. In this work, the sequence of the mitochondrial cytochrome oxidase subunit I region was examined in individuals of these species to identify restriction site variation and develop polymerase chain reaction-restriction fragment length polymorphisms assays. Species-specific restriction endonuclease patterns were found with the enzymes AluI, HaeIII and MspI, allowing an exact identification of Donax species. This methodology provides simple, reliable and cost-effective identification of four Donax species and may be useful to prevent commercial fraud and to increase food traceability.