The variability of the protein expression profiling in the extracellular products (ECPs) of in vitro cultured Perkinsus olseni deriving from 4 regions of the Spanish coast was evaluated. The regions involved were the rías of Arousa and Pontevedra (Galicia, NW Spain), Carreras River (Andalusia, SW Spain) and Delta de l'Ebre (Catalonia, NE Spain). P. olseni in vitro clonal cultures were produced from parasite isolates from four clams from each region. Proteins released by the in vitro cultured parasites were isolated and separated by two dimensional electrophoresis (2DE). Qualitative comparison of protein expression profiles in the P. olseni ECPs among clones from all the regions was performed with PD Quest software. Around 130 spots were counted in the gels from ECPs of P. olseni clones from each region, of which 23 spots were shared by clones from all the regions and various spots were representative from clones of one region (appear in every clonal culture from that region but did not in every one of the other regions). A total of 34 spots were excised from the gels and analysed for sequencing. The protein cathepsin B, involved in proteolysis, the signal recognition particle receptor subunit β, involved in protein transport through membranes, and a protein belonging to N-acetyl transferase superfamily, involved in biosynthesis, were identified in spots shared by P. olseni ECPs from all regions. Pepsin A precursor, involved in proteolysis; heat shock protein (HSP) 60; and phosphoserine aminotransferase, involved in biosynthesis, were representative of P. olseni ECPs from Ría de Arousa, while peroxiredoxin V, involved in oxidation-reduction, was representative of P. olseni ECPs from Ría de Pontevedra. Differences in released proteins suggest different virulence or resistance to host attack between parasites from different locations.