The genetic differentiation of Solea solea and Solea senegalensis from several estuarine systems along the Portuguese coast was studied. Nine polymorphic isozyme loci (ACP-1*, ACP-2*, GPI-1*, GPI-2*, sMDH*, ME-1*, ME-2*, MPI* and PGM*) were analysed using starch gel electrophoresis and isoelectric focusing. Differentiation between the species was high (mean average Nei distance of 0.93). The most efficient loci in the diagnosis of the two species were ACP-1*, ME-2* and GPI-2*. S. solea showed a higher genetic diversity than S. senegalensis. Within each species a low genetic differentiation between the samples analysed was found. Although with a low magnitude the interpopulational genetic differentiation of S. solea was higher than that of S. senegalensis. This could probably be explained by some particularities of the life cycles of these species, namely the more extended period of occurrence of larval stages of S. senegalensis in the plankton. Although no clear evidence about the population structure model emerged from the analysis of several Atlantic and Mediterranean populations of S. solea, the significant correlations obtained between genetic and geographical distances support an isolation by distance model.