The Mediterranean mussel (Mytilus galloprovincialis) is widely used in monitoring programs and in ecotoxicological studies to examine the biological effects of physicochemical parameter changes and the impact of chemical pollutants. Metabolomics has recently demonstrated high potential to gain further insight into the molecular effects of chemical exposure and the success of its application is dependent on the extent of prior metabolomics knowledge available on the target organism. Therefore, the purpose of this study was the investigation of the metabolites of five different functional tissues of male and female Mediterranean mussels (digestive gland, foot, gill and gonad tissues and in the remaining soft tissues) accessible to the analysis using the most common sample preparation recommended for tissue analysis (i.e. Bligh & Dyer). Metabolic fingerprints were acquired via liquid chromatography high-resolution mass spectrometry and the identification was based on an internal database developed in the laboratory. It led to the identification of 110 metabolites, among which amino acids, carboxylic acids, purine and pyrimidine metabolites were often the most abundant. The metabolic contents of the five tissues quantitatively and qualitatively differed, with a clear distinction between male and female contents observed in the gonads and digestive glands. These results underline the importance of selecting the most suitable tissue and sex to study the impact of contamination on metabolism and the need for further research to deeper characterize the metabolome of this organism.