The 45S and 5S rDNA loci were physically mapped by double color fluorescent in situ hybridization (FISH) on the mitotic metaphase chromosomes of F. esculentum and F. tataricum. The results showed four pairs of 45S rDNA loci on ⅠS, ⅡS, ⅢL, and ⅤL (L and S represent chromosome long and short arm respectively, and the Roman number indicate chromosomes serial number, hereinafter the same), as well as two pairs of 5S rDNA loci on ⅠL and ⅣS in F. esculentum, and five pairs of 45S rDNA loci on ⅠS, ⅡS, ⅢL, ⅤL, and ⅦS, as well as three pairs of 5S rDNA loci on ⅠL, ⅣS, and ⅥS in F. tataricum. There are obviously differences in 45S and 5S rDNA loci of F. esculentum and F. tataricum, suggesting they are far in phylogenies. Based on 45S and 5S rDNA loci location and karyotype characteristics of the mitotic metaphase chromosomes, eight pairs of homologous chromosomes of F. esculentum and F. tataricum all can be distinguished distinctly, respectively.