The karyotype of the rainbow trout is characterized by a primitive XX/XY sex-determining chromosomal system. (Thorgaard et al., 1977). In the present study using FISH we have physically linked the 5S rRNA genes to the partially undifferentiated X chromosome pair. PCR amplified 5S rDNA was used for FISH and hybridization signals indicated that the genes were duplicated, present in one acrocentric and one metacentric pair of chromosomes. After analyzing several individuals, the female metaphases showed four fluorescent signals whereas males presented only three signals. Two of the three signals obtained in males corresponded to the metacentric pair whereas the single signal was mapped to the heterochromatin that cytologically differentiates the X chromosome from the Y chromosome. Double FISH experiments demonstrated that the 5S rDNA which is not sex linked is located at the NOR bearing arm close to the major ribosomal RNA genes (5.8S, 18S and 28S), similar to the situation observed in Atlantic salmon (Pendas et al., 1994a).