The genetic relationships between several wild species and subspecies of the genus Hordeum were assessed using fluorescence in situ hybridization (FISH). Plant material included natural populations of wild barley growing in Spain of the annual species, H. marinum ssp. marinum (2n = 14) and gussoneanum (2n = 14), and H. murinum ssp. murinum (2n = 28), and leporinum (2n = 28) and the perennial species H. bulbosum (2n = 14) and H. secalinum (2n = 28), plus the South American perennial species H. chilense (2n = 14). FISH was used to locate the chromosomal sites of two rDNA multigene families 5S and 18S-26S (pTa71 and pTa794) and three repetitive DNA sequences (pSc119.2, pAs1 and pHch950) isolated from different species and genera. The seven chromosomes of the diploid species were readily distinguished by their external morphology and hybridization patterns to pTa71, pTa794, pSc119.2 and pAs1. These DNA probes were also useful for the identification of homologous chromosomes and in differentiating these from unidentified chromosomes in the tetraploid taxa. The use of the probe pHch950 permitted intergenomic differentiation in tetraploids and supports the diphyletic origin of H. murinum and H. secalinum. The in situ experiments yielded the following conclusions: (1) differences between the sub-species marinum and gussoneanum; (2) close relationships between the subspecies murinum and leporinum; and (3) major differences in physical mapping between H. bulbosum and the remaining taxa. The genomic and phylogenetic relationships between taxa, as inferred from the results, are discussed.