Fluorescence in situ hybridization (FISH) is a powerful tool for the detection of DNA sequences in the specific region of the chromosomes. As well as for the integrated physical mapping, FISH karyotype analysis has to be preceded. The detailed karyotypes of two onion cultivars, which are resources for onion genome sequencing project (‘Eumginara’ and ‘Sinsunhwang’), were constructed based on triple color fluorescence in situ hybridization (FISH) using 5S rDNA, 45S rDNA, and tandem repeat sequence. All used our materials showed 2n=2x=16 with x=8 as basic chromosome number. 5S rDNAs were located on 4 loci in one pair of interstitial region of short arm chromosome in both onion cultivars. Two pairs of 45S rDNAs were positioned in distal region of short arm chromosome in ‘Eumginara’. Otherwise 5 loci of 45S rDNAs were located in distal region of two pairs of short arm chromosome in ‘Sinsunhwang’. Among them, two signals of 45S rDNAs were co-localized in distal part of short arm and long arm chromosome, respectively. In case of tandem repeat sequence was detected on telomeric region of 8 pairs of chromosomes except on 45S ribosomal DNA sites. These results will provide a valuable background for physical mapping and help to further more understand the genome sequencing project in Allium cepa.