Populations of the flounder, Platichthys flesus, were screened for electrophoretically detectable protein variation at up to 37 loci. Atlantic and North Sea populations (subspecies flesus) were genetically very similar to one another (I>0.99) but different from Adriatic (subspecies italicus) and Black Sea (subspecies luscus) populations. The values for genetic identity between subspecies were around 0.9. Diagnostic loci enabled specimens of flesus, italicus and luscus to be differentiated from one another. Samples of flesus showed two to three times the heterozygosity levels ofitalicus or luscus, consistent with the greater population size offlesus. Morphological comparisons enabled populations to be categorized to subspecies. Platichthys flesus italicus and luscus are valid subspecies: the terms are not synonyms. The genetic distance data provide an estimate of divergence time of the three subspecies at about 2 My ago, an estimate that seems reasonable in the light of present knowledge concerning the recent geological histories of the Mediterranean and Black Sea basins.