The genetic structure of European flounder Platichthys flesus populations was analysed in the southern part of the species' distribution area, from England to Portugal; samples were collected in 5 estuaries which were chosen because of their latitudinal cline, the level of contaminants and the possibility of hypoxia: the Tamar in England; the Canche, Seine and Vilaine in France; and the Mondego in Portugal. Analysing the variability of a mitochondrial marker (cytochrome c oxidase subunit 1), we observed (1) a moderately higher nucleotide diversity in the Tamar and Canche, reflecting the probable existence of a major refugium in this sector during the last glacial maximum, and (2) a reduced genetic differentiation over the whole data set. The use of 8 microsatellites underlined the maintenance of genetic diversity in the peripheral population of the Mondego, located near the southern limit of the species' range; furthermore, significant pairwise differentiations of populations including Mondego underlined the particular genetic status of this Portuguese population. The polymorphism of a candidate gene involved in energetic metabolism, AMP-desaminase isoform 1, was investigated focusing on exon 1; we found the highest heterozygosities in the more polluted populations (Seine, Tamar) and marked differentiations compared to the other group of populations (Mondego, Vilaine, Canche). The pattern of variability observed for the AMP-desaminase suggests that in chronically contaminated flounder populations, cocktails of pollutants act as selective agents directly or indirectly on this locus.