Comparisons of the levels of genetic variation within and between a hatchery F-1 (FAR, n=116) of Senegalese sole, Solea senegalensis, and its wild donor population (ATL, n=26), both native to the SW Atlantic coast of the Iberian peninsula, as well as between the wild donor population and a wild western Mediterranean sample (MED, n=18), were carried out by characterizing 412 base pairs of the nucleotide sequence of the mitochondrial DNA control region I, and six polymorphic microsatellite loci. FAR showed a substantial loss of genetic variability (haplotypic diversity, h=0.49 +/- 0.066; nucleotide diversity, pi=0.006 +/- 0.004; private allelic richness, pAg=0.28) to its donor population ATL (h=0.69 +/- 0.114; pi=0.009 +/- 0.006; pAg=1.21). Pairwise F-ST values of microsatellite data were highly significant (P<0.0001) between FAR and ATL (0.053) and FAR and MED (0.055). The comparison of wild samples revealed higher values of genetic variability in MED than in ATL, but only with mtDNA CR-I sequence data (h=0.948 +/- 0.033; pi=0.030 +/- 0.016). However, pairwise Phi(ST) and F-ST values between ATL and MED were highly significant (P<0.0001) with mtDNA CR-I (0.228) and with microsatellite data (0.095), respectively. While loss of genetic variability in FAR could be associated with the sampling error when the broodstock was established, the results of parental and sibship inference suggest that most of these losses can be attributed to a high variance in reproductive success among members of the broodstock, particularly among females.