Using the fluorescence in situ hybridization (FISH) technique, we conducted karyotype analyses to identify the lost chromosomes us three somaclonal variants obtained from tissue culture of wild Allium tuherosum (2n = 4X = 32). The three lost chromosomes of the At29 variant (2n = 29) were all chromosome 2, the two for At30 (2n = 30) were chromosomes 7 and 8, and At31 was missing chromosome 2. Chromosome compositions of these variants were confirmed as being fixed lines during two years of greenhouse cultivation. The bicolor FISH technique, involving both 55 and 185-5.8S.26s ribosomal RNA genes as probes, was used to assign rhromosomal locations and to confirm whether the lost chromosomes contained any rRNA markers. The 5S rRNA gene signals in all variants au well as the wild type were detected as two sets, one on the intercalary region of the short arm of chromosome 3, the other on the intercalary region of the long arm of chromosome 6. One 185-5.8S-26S rRNA gene site on the secondary constriction included a flanking satellite and terminal region on the short arm of chromosome 8. Signals of the 185-5.9S-26S rRNA gene in At3O showed in only three chromosomes, indicating that one of the lost chromosomes was chromosome 8. Overall, three marker chromosomes were established by FISH, using rRNA multigene families.