Seafood has been identified as the food commodity with the second highest potential for fraud by the European Parliament. Thus, the objective of this project is the development and validation of a DNA metabarcoding method for the identification of seafood in processed foods.Reference sequences of the mitochondrial 16S rDNA marker gene were obtained from the National Centre for Biotechnology. We designed universal primers targeting fragments of 150 bp to 210 bp, and PCR conditions were established. Reference material (202 samples), DNA extract mixtures and processed foods (112 samples) were sequenced on Illumina platforms. Automized data evaluation was achieved by creating customized databases in combination with a Galaxy analysis pipeline. The DNA metabarcoding method was successfully developed and validated. Five primer systems were created that amplify the barcodes of mussels, razor clams, sheath clams, venerids and cockles, oysters and scallops, squids, crustaceans, or marine and terrestrial snails. We correctly identified seafood in reference material, model food samples and processed foods with complex matrices such as prawn butter, linguine with squid ink and nutritional supplements. Most specimens can be classified at species or genus level. The heat stability of the DNA provides an advantage in the analysis of commercial foods. Additionally, this untargeted, standardized method allows the screening of species in complex mixtures, unlike species-specific PCR and DNA barcoding. Establishing this method in official control laboratories directly contributes to improving consumer protection by ensuring the authenticity and safety of food.