Glutathione peroxidases are a family of antioxidant enzymes catalyzing the reduction of H2O2 or organic hydroperoxides. In the present study, we report the molecular characterization and gene expression analysis of a new GPx-1 from the Antarctic fish Trematomus bernacchii. To expand our knowledge on the GPx-1 s evolution within the group of Antarctic fish, in this work, we also presented the cDNA sequencing of this enzyme in other three species, belonging to two families—Nototheniidae (Trematomus eulepidotus, T. lepidorhinus) and Bathydraconidae (Cygnodraco mawsoni). The deduced amino acid sequences were compared with GPx-1 s of other vertebrates by multiple alignment, in order to evaluate the conservation of amino acids involved in the enzyme activity. The results of phylogenetic analyses indicated that fish GPx-1 s possibly originated from independent duplication events, and Antarctic GPx-1 s evolved according to the molecular and morphological phylogeny of Antarctic fish. Basal GPx-1 mRNA expression analyses in various tissues of T. bernacchii specimens indicated that liver and heart displayed the highest mRNA accumulation; probably a protection of these organs against lipid peroxidation is needed.