By analyzing three multigene families, two closely related and commercially important species, Dicentrarchus labrax and Dicentrarchus punctatus, were characterized by cytogenetic and molecular methods. The interspecies hybrid Dicentrarchus labrax (♀) × Dicentrarchus punctatus (♂) was also analyzed. The multigene families studied were the 5S rDNA, 45S rDNA and the U2 snRNA. A microsatellite GTT motif was found within the non transcribed spacers (NTS) of the 5S rDNA from the two species. However, hexanucleotide duplication next to this microsatellite was observed in the D. labrax and hybrid clones, but not in D. punctatus. The U2 snRNA appeared to be linked to the U5 gene and showed two variant sequences, in both D. labrax and D. punctatus. They differed in one insertion/deletion of 7 nucleotides. The first internal transcribed spacer (ITS-1) region showed higher nucleotide variability in D. punctatus than in D. labrax. Nucleotide polymorphism within species and also nucleotide divergence between species were determined in the different gene regions. In a FISH analysis we obtained three chromosomal markers, because the 5S rDNA, 18S rDNA and U2 snRNA probes hybridized each in three different chromosome pairs. Hence none of them was co-localized. The 5S rDNA cluster and U2 snRNA were localized in acrocentric chromosome pairs, while the 18S rRNA gene probe hybridized in a subtelocentric pair. Finally, the usefulness of the results in developing tools for phylogenetic analysis and species identification are discussed in relation to other fish species.