Physical mapping of the 5S rDNA loci and 45S rDNA loci was carried out in eleven different Hordeum species by double fluorscent in situ hybridization. The number of loci varied from one (H. bulbosum and H. erectifolium) to seven (H. marinum) for 5S rDN and from one (H. marinum) to seven (H. brachyantherum) for 45S rDNA depending on species. In some species, two 5S rRNA gene loci were found in the same chromosome arm (H. chilense, H.jubatum, H. lechleri, H. marinum, H. brachyantherum, and H. depressum). However, none of the species had duplicate 45S rRNA gene loci in the same chromosome arm. H. carpense had a chromosome with two 5S rRNA gene loci in a chromosome in both arms, while H. brachyantherum had a chromosome with a 45S locus in both arms. Many cases were found with a 5S rDNA locus and a 45S rDNA locus at the same chromosomal site or in close proximity (H. chilense, H. jubatum, H. procerum, H. lechleri, H. marinum, H. brachyantherum, H. depressum, and H. erectifolium). Although major rRNA gene loci were found to be conserved among species in a few cases, minor loci were variable in terms of their chromosomal locations, which prevents our getting a clear picture on the karyotype evolution of the rDNA bearing chromosomes. The possibility of segmental polyploidy is proposed to explain the observed minor rDNA loci variations.