5S ribosomal gene spacer sequences from the short-spacer arrays of wheat and rye were isolated by PCR. The 29 new DNA sequences displayed noticeable heterogeneity at scattered positions. Nevertheless, based on shared DNA sequence polymorphisms, sequence alignment clearly classified the sequences into three groups. Group-specific primer sets were designed to allow chromosomal assignment by PCR on nullitetrasomic wheat stocks, as well as on wheat-rye translocation and addition lines. The three groups were assigned to orthologous loci 5S-Rrna-B1, 5S-Rrna-D1, and 5S-Rrna-R1 on homoeologous chromosomes 1B, 1D, and 1R, respectively. Hence, group-specific DNA sequence variation could be related to fixed orthologous DNA sequence variation between 5S rRNA multigene families on the homoeologous group 1 chromosomes. In addition, members of the three groups showed fixed orthologous length polymorphism. Four sequenced 5S-Rrna-B1 units, however, had a duplication in the gene encoding region and are probably representatives of a nontranscribed subfamily of 5S rDNA repeating units. The observed chromosome-specific polymorphisms among sequences belonging to a multigene family with thousands of copies suggests that this type of polymorphism may exist in many genes and gene families in polyploid wheats. The implication of this finding in relation to the construction of molecular tools for wheat-genome analysis and manipulation is discussed.