The chromosomal locations of the 18S-26S (45S) and 5S rDNA loci in cytotypes AA, BB, and AABB of Scilla scilloides Complex from Korea were physically mapped using multicolor fluorescence in situ hybridization (McFISH). Genomic in situ hybridization (GISH) was also performed to distinguish between the AA and BB genomes in allotetraploid AABB plants. One 18S-26S rDNA locus was detected in both AA (a2) and BB (b1); one locus also was found in the allopolyploid AABB (b1). This demonstrated the loss of that locus in genome A. GISH with biotin-labeled DNA from the BB genome and digoxigenin-labeled 18S-26S rDNA probes revealed that the 18S-26S rDNA in AABB plants was localized in the nucleolus organizer region (NOR) of genome B. One and two 5S rDNA loci were found in diploids AA and BB, respectively. As expected, all three 5S rDNA loci were detected in the AABB plants. The sequence identities of the 55 rDNA genes among cytotypes AA and BB, AA and AABB, and BB and AABB were 99%, 95%, and 95%, respectively.