Sequential genomic in situ hybridization (GISH) and double color fluorescence in situ hybridization (FISH) techniques were used to map physically 45S rDNA and 5S rDNA genes in a wheat-H. villosa 6V/6A substitution line. The results indicated that eight pairs of 45S rDNA loci were detected on seven pairs of wheat chromosomes, and six pairs of 5S rDNA loci were detected on six pairs of wheat chromosomes, respectively. Both 45S and 5S rDNA signals were collocated closely on the terminal region of the short arm of chromosomes 1A, 1B and 5D. At the same time, we discussed the possible factors of variation of the two types of rDNA in locus number and location in wheat-H. villosa 6V/6A substitution line.