The Merluccidae family comprises marine species, some of them of high commercial value and others less appreciated, whose commercialization in Europe under the generic name of "hake" is highly remarkable. The potential of proteomics was employed in this study with the aim of achieving the differential characterization of five different hake species: Merluccius merluccius (European hake), M. australis (Southern hake), M. hubbsi (Argentinian hake), M. gayi (Chilean hake), and M. capensis (Cape hake), some of them very closely related. Species-specific polypeptides were observed for the five hake species studied in isoelectric focusing (IEF) and/or two-dimensional electrophoresis (2-DE) high-resolution gels. The peptide mass maps of two polypeptide groups, previously selected by 2-DE analysis as potentially species-specific, were obtained by "in-gel" tryptic digestion, followed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Analysis of group A polypeptides (with pi in the range of 5.0-5.5 and molecular mass of 17 kDa), allowed the differential classification of the hake species into two groups: the East Atlantic coast group and the West Atlantic coast group. Moreover, the peptide mass-maps from the heat-resistant parvalbumin fraction (pl below 4.5; molecular mass < 12 kDa) allowed the detection of a peptide characteristic of M. australis not present in the other four hake species tested. A specific 17 kDa protein from M, merluccius was also partially sequenced by nanospray-ion trap-tandem MS, revealing a high homology with rat nucleoside diphosphate kinase A (NDKA). This work opens the way to the application of proteomics to the differential characterization of commercial hake species at the molecular level.