This study was designed to characterize the 5S ribosomal DNA repeat unit and to evaluate its phylogenetic informativeness in Mimachlamys varia, Hinnites distortus, Aequipecten opercularis and Pecten maximus, scallops of the bivalve family Pectinidae. The repeat unit was PCR amplified and several products cloned and sequenced. The deduced coding region covered 120 bp, showed 52-53% GC content and an identifiable internal promoter. The spacer region was 313-345 bp in length with 30-40% GC and the ends contained elements showing similarity with upstream and downstream sequences involved in the transcription. The sequences of P. maximus were identical and those of M. varia and H. distortus differed only at 2-3 positions. However, the sequences of A. opercularis showed a percentage of differences of 0.69-9.15%, distinguishing two types of units differing in sequence and length of the spacer region. All scallops displayed an identical gene sequence, except M. varia that differed by one nucleotide substitution, but a highly variable spacer. In the phylogenetic trees the sequences grouped by species with two well supported clades, one containing the sequences of M. varia and H. distortus and the other those of P. maximus and A. opercularis, the latter grouped according to the unit type. The topology obtained was in accordance with scallop evolutionary relationships inferred from previous phylogenetic studies.