It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta. In regard to the usual two positions of linked 5S rDNA to the 3 ' end of 25S rDNA, two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica, one of species in Ochrophyta. This result supplemented the previous report that 5S rDNA was unlinked to 25S rDNA in this kelp. In order to simultaneously visualize this unlinkage of two genes, dual-color fluorescence in situ hybridization (FISH) technique was applied to the cytogenetics of S. japonica. Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte, respectively, metaphase nuclei hybridized simultaneously with the labeled probes of 18S rDNA and 5S rDNA. Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18S-5.8S-25S rDNA was localized at the interstitial region of Chromosome 23, whereas 5S rDNA resided at the sub-telomeric region of Chromosome 27. These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene, and their loci on different chromosomes indicated the physical unlinkage of 5S rDNA to 18S-5.8S-25S rDNA in this kelp. Therefore, dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S. japonica.