The 18S–5.8S–26S rRNA genes have been located on five pairs of barley (Hordeum vulgare L., 2n = 2x = 14) chromosomes (in descending order of copy number, barley chromosome numbers 6, 7, 5, 1, and 2; homoeologous groups 6I, 5I, 1I, 7I, and 2I) by in situ hybridization followed by C-banding. All sites were at intercalary positions. The pairs of major sites on chromosomes 6 (6I)1 and 7 (5I) are well known. Silver staining of nuclei and meiotic analysis have previously indicated that additional rDNA sites may be present, but the presence of sites on a further three chromosome pairs was unexpected. Within the tribe Triticeae, few species have more than two pairs of rDNA sites, and they have not been reported on group 2 chromosomes. We propose calling the new sites Nor-I1 (on chromosome 5 (1I)), Nor-I4 (on chromosome 1 (7I)), and Nor-I5 (on chromosome 2 (2I)), and that any further rDNA sites on homoeologous group 2 chromosomes should be called Nor-5. As conventional, all designations are based on temporal order of discovery in the Triticeae and designating the H. vulgare genome as I. In situ hybridization is valuable for gene mapping, since it can detect the presence of genes with a very wide range of copy numbers at different sites.Key words: C-banding, nucleolus organizer regions, fluorescent in situ hybridization, Hordeum vulgare, gene mapping.