5S ribosomal DNA (rDNA) sequences were analyzed in four species belonging to different genera of the fish family Batrachoididae. Several 5S rDNA variants differing in their non-transcribed spacers (NTSs) were found and were grouped into two main types. Two species showed both types of 5S rDNA, whereas the other two species showed only one type. One type of NTS of Amphichthys cryptocentrus showed a high polymorphism due to several deletions and insertions, and phylogenetic analysis showed a between-species clustering of this type of NTS in Amphichthys cryptocentrus. These results suggest a clear differentiation in the model of 5S rDNA evolution of these four species of Batrachoididae, which appear to have been subject to processes of concerted evolution and birth-and-death evolution with purifying selection.