The essential trace element selenium is important for human health, but selenium deficiency or oversupply can also cause diseases. Therefore, selenium uptake through diet is of great importance. It depends on selenium concentrations in foodstuff, seleniums bioavailability to the body, as well as its conversion to metabolically active forms. Since bioavailability is influenced by the form of selenium present in foodstuff, total selenium together with selenium speciation has to be determined.Fish can be an important source of selenium. In this work, muscle of tuna, mackerel, and sardine were investigated for their selenium speciation. Selenoneine (2-selenyl-N, N, N-trimethyl-L-histidine), the selenium analogue of ergothioneine, was the major water extractable selenium species. In tuna, a selenosugar and Se-methylselenoneine were for the first time identified as minor selenium species.Due to its proposed antioxidant activity and possible role in mercury detoxification, selenoneine might be of interest to human health. Hence, sample storage and preparation for its quantitative determination in tuna muscle was investigated. Extraction with 1,4-dithiothreitol (DTT) resulted in better extraction efficiencies than water extraction, especially when samples had been stored frozen before extraction. About 40% of the total selenium was solubilized by DTT, with selenoneine accounting for about 50% of the extracted selenium in tuna muscle. Storage of tuna muscle at room temperature after freeze-drying or frozen at -20C showed decreases in extractable selenoneine. Therefore, determination of the target compound should be performed on fresh samples. When selenoneine in fish extracts has to be preserved for later determination, stabilization through reduction with DTT and subsequent derivatization with iodoacetamide followed by storage at -20C was found most suitable. The results of this study are going to advance the reliable quantification of selenoneine in foodstuff.