An accurate physical map of the localization of the 5S and the 118S-26S rRNA genes has been constructed by bi-color fluorescence in situ hybridization in Allium wakegi cultivar and its regenerant (AW 02-18). A. wakegi cultivar is an amphihaploid species with 16 chromosomes consisting of two different genomes. A rhodamine-labeled 5S rDNA and a biotin labeled 18S-26S rDNA were used as probes for this study. Probe DNAs were labeled by nick translation method. The number of 5S rDNA regions were three on chromosomes 9 and 15, and those of 18S-26S rDNA region were three on chromosomes 6, 10 and 14. The signals of 5S rDNA were detected on the intercalary region of short arm in chromosome 15, and two regions of short arm in chromosome 9. The signals of 18S-26S rDNA were detected on terminal region of short arm and a part of the satellite containing secondary constriction region in chromosome 6, terminal region of short arm in chromosome 10, and terminal site containing satellite chromosome and secondary constriction region in chromosome 14. While the 18S-26S rDNA sites were corresponded to C-bands, 5S rDNA were not. In a AW 02-18 amphidiploid regenerant, homologous chromosomes were exactly identified by chromosomal localization of rRNA gene families through ISH. The technique of bi-color fluorescence in situ hybridization in Allium species is the first reported through this work.