Two loci containing genes encoding 5S rRNA were mapped on the second and third chromosomes of barley. The two gene clusters located on different chromosomes differed in the length of the nontranscribed spacer separating the 5S rRNA genes. All nontranscribed spacers contained a variable number of trinucleotide tandem repeats. The distribution of 5S genes between these two clusters and their copy number varied widely between cultivars and doubled haploids derived from a cross between two barley cultivars. However, this variation had no obvious effect on plant phenotype.