The 5S rRNA gene is considered as valuable resource for chromosome landmarks and evolutionary studies. In this study, the tandem repeat unit of 5S rRNA gene containing the coding sequence and non‐transcribed spacer (NTS) region was amplified by PCR, cloned, and sequenced from Allium wakegi (2n=XY=16) and the two ancestors, A. cepa and A. fistulosum. Although there were intraspecific variations among the clones of each species, we could construct each consensus sequences for A. cepa and A. fistulosum and two consensus sequences for A. wakegi. The sequence of the 120 bp coding region was completely homologous among the consensus sequences from the Allium species examined. However, the sequence in the NTS region was significantly variable and informative in genome analysis. The variations were found to be clustered between the positions 230 and 269, and also distributed broadly as single nucleotide. A. wakegi could be divided into two classes by the 5S rRNA sequences (AWAK‐1 and AWAK‐2). AWAK‐1 showed high homology to the genome of A. cepa and AWAK‐2 to that of A. fistulosum. Fluorescence in situ hybridization technique was applied to analyze the distribution of 5S gene loci. In A. wakegi, 5S rRNA sequences were detected in two different chromosomes, each one showing the pattern identical to the chromosome donated from each genome of both ancestors.