The potential of Raman spectroscopy was investigated in terms of its capability to discriminate the species of the fish samples and determine their freshness according to the number of freezing/thawing cycles they exposed. Species discrimination analysis was carried out on sixty-four fish samples from six different species, namely horse mackerel (Trachurus trachurus), European anchovy (Engraulis encrasicolus), red mullet (Mullus surmuletus), Bluefish (Pomatamus saltatrix), Atlantic salmon (Salmo salar) and flying gurnard (Trigla lucerna). Afterwards, fish samples were exposed to different numbers of freezing/thawing cycles and separated into three batches, namely (i) fresh, (ii) once frozen-thawed (OF) and (iii) twice frozen-thawed (TF) samples, in order to perform the freshness analysis. Raman data collected were used as inputs for chemometric analysis, which enabled us to develop two main PCA models to successfully terminate the studies for both species discrimination and freshness determination analysis. (C) 2014 Elsevier Ltd. All rights reserved.