Four minor rDNA loci have been mapped physically to barley (Hordeum vulgare L.) chromosomes 1 (7I), 2 (2I), 4 (4I), and 5 (1I) by a two-step in situ hybridization procedure including a GAA microsatellite sequence. Reprobing with the microsatellite resulted in a distinct banding pattern, resembling the C-banding pattern, which enabled unequivocal chromosome identification. This study suggests that gene mapping accuracy may be improved by using probes with well-characterized and narrow hybridization sites as cytological markers which are situated close to the gene locus. One of the rDNA loci is located about 54% out on the short arm of chromosome 4 and it has not previously been reported in barley. We have disignated the new locus Nor-16. rDNA loci on homoeologous group 4 chromosomes have not yet been reported in other Triticeae species. The origin of these 4 minor rDNA loci is discussed in relation to their equilocal distribution on the chromosomes.