Non-denaturing FISH (ND-FISH) was used to compare the distribution of four simple sequence repeats (SSRs)-(AG) n , (AAG) n , (ACT) n and (ATC) n -in somatic root tip metaphase spreads of 12 barley (H. vulgare ssp. vulgare) cultivars, seven lines of their wild progenitor H. vulgare ssp. spontaneum, and four lines of their close relative H. bulbosum, to determine whether the range of molecular diversity shown by these highly polymorphic sequences is reflected at the chromosome level. In both, the cultivated and wild barleys, clusters of AG and ATC repeats were invariant. In contrast, clusters of AAG and ACT showed polymorphism. Karyotypes were prepared after the identification of their seven pairs of homologous chromosomes. Variation between these homologues was only observed in one wild accession that showed the segregation of a reciprocal translocation involving chromosomes 5H and 7H. The two subspecies of H. vulgare analysed were no different in terms of their SSRs. Only AAG repeats were found clustered strongly on the chromosomes of all lines of H. bulbosum examined. Wide variation was seen between homologous chromosomes within and across these lines. These results are the first to provide insight into the cytogenetic diversity of SSRs in barley and its closest relatives. Differences in the abundance and distribution of each SSR analysed, between H. vulgare and H. bulbosum, suggest that these species do not share the same H genome, and support the idea that these species are not very closely related. Southern blotting experiments revealed the complex organization of these SSRs, supporting the findings made with ND-FISH.