Sets of microsatellites extracted from both a genomic library (gSSRs) and from expressed sequence tag sequence (eSSRs), and single nucleotide polymorphisms (SNPs) were applied to assess the levels of genetic diversity in a sample of 70 barley accessions, originating from 28 countries in Asia, Africa, the Middle East and Europe. The eSSR assays detected a mean of 9.5 alleles per locus, and the gSSRs only 5.7 alleles per locus, but the polymorphism information content values for the two assay types were indistinguishable. Strong and statistically significant correlations were observed between the eSSR and gSSR (r = 0.86, P < 0.05), the eSSR and SNP (r = 0.74, P < 0.05) and the gSSR and SNP genotypes (r = 0.67, P < 0.05). Accessions originating from the Middle East and Asia had the highest levels of genetic diversity. Pairwise genetic similarity ranged from 0.16 to 0.87 (mean 0.43), indicating that the sample was genetically diverse. When clustered on the basis of genotype, Asian and African accessions tended to be grouped together, but those originating from the Middle East were not concentrated in any particular cluster.