The 5S rRNA gene in higher eukaryotes is organized into repeated units of tandem array that comprise a conserved 120-bp coding region and a non-transcribed spacer (NTS) of variable length with nucleotides. The allotetraploid genome of Allium sacculiferum consists of two unknown diploids (2n=32). Analyses have not been successful toward clarifying the origin of each genome due to their similar chromosome morphology and unmatched C-banding patterns. We PCR-amplified the coding and NTS regions of its 5S rRNA genes, cloned them into vectors, and determined their DNA sequences. Interestingly, the aligned sequences of the NTS clones could be divided into two distinctive groups based on the existence of a 3-bp CCT insertion/deletion at the beginning of the NTS region. This feature makes it an important genetic marker for distinguishing the origin of the A. sacculiferum chromosomes. Furthermore, by applying fluorescence in situ hybridization, we located the 5S rRNA gene loci on Chromosomes 5, 7, 8, 9, and 14; their distribution is unique to A. sacculiferum. These data support the idea that one set of this genome has originated from a CCT-containing close relative -- A. deltoid-fistulosum -- and that the NTS region may be used as a molecular marker for identifying parental lines for the allotetraploidity of A. sacculiferum.