Using fluorescence in situ hybridization (FISH), 5S rDNA gene was mapped on chromosomes of half-smooth tongue-sole(Cynoglossus semilaevis)and olive flounder(Paralichthys olivaceus)for the first time. The results showed that 5S rDNA has two hybridization signal loci in a pair of homologous chromosomes of male and female half-smooth tongue-sole, respectively; 5S rDNA has two and three hybridization signal loci in a pair of homologous chromosomes of diploid and triploid olive flounder, respectively, referring to the clear and specific hybridization signals. FISH mapping of 5S rDNA genes on the chromosomes of diploid half-smooth tongue-sole and triploid olive flounder provides a feasible method for ploidy and chromosome identification of half-smooth tongue-sole and olive flounder. In addition, the coding region sequences of 5S rDNA gene of turbot (Scophthalmus maximus) and half-smooth tongue-sole were amplified using same primers designed for olive flounder 5 S rDNA gene, and the phylogenetic analysis of the 5S rDNA genes in four species of flatfishes, including plaice(Pleuronectes platessa), senegalese sole (Solea senegalensis), largemouth bass(Micropterus salmoides) and arctic lamprey(Lampetra japonica) showed that the 5S rDNA gene of five species of flatfishes shared high homology (98.3%) and gathered into a branch. The GC content was significantly higher than the AT content in conservative sequence of 5S rDNA coding region of these five species of flatfishes.