We evaluated the proteome variability of in vitro-cultured Perkinsus olseni cells deriving from 4 regions of the Spanish coast: the rías of Arousa and Pontevedra (Galicia, NW Spain), Carreras River in Huelva (Andalusia, SW Spain) and Delta de l'Ebre (Catalonia, NE Spain). P. olseni in vitro clonal cultures were produced starting from parasite isolates from 4 individual clams from each region. Those clonal cultures were used to extract cell proteins, which were separated by 2-dimensional (2D) electrophoresis. Qualitative comparison of P. olseni protein expression profiles among regions was performed with PD Quest software. Around 700 protein spots from parasites derived from each region were considered, from which 141 spots were shared by all the regions. Various spots were found to be exclusive to each region. Higher similarity was found among the proteomes of P. olseni from the Atlantic regions than between those from the Mediterranean and the Atlantic. A total of 54 spots were excised from the gels and sequenced. Nineteen proteins were annotated after searching in databases, 13 being shared by all the regions and 6 exclusive to 1 region. Most of the identified proteins were involved in glycolysis, oxidation/reduction, metabolism and response to stress. No direct evidence of P. olseni variability associated with virulence was found within the protein set analysed, although the differences in metabolic adaptation and stress response could be connected to pathogenicity.