Due to the availability of sequence data from large-scale EST (expressed sequence tag) projects, it has become feasible to develop microsatellite or simple sequence repeat (SSR) markers from genes. A set of 111 090 barley ESTs (corresponding to 55.9 Mb of sequence) was employed for the identification of microsatellites with the help of a PERL5 script called MISA. As a result, a total of 9 564 microsatellites were identified in 8 766 ESTs (SSR-ESTs). Cluster analysis revealed the presence of 2 823 non-redundant SSR-ESTs in this set. From these 754 primer pairs were designed and analysed in a set of seven genotypes including the parents of three mapping populations. Finally, 185 microsatellite (EST-SSRs) loci were placed onto the barley genetic map. These markers show a uniform distribution on all the linkage groups ranging from 21 markers (on 7H) to 35 markers (3H). The polymorphism information content (PIC) for the developed markers ranged from 0.24 to 0.78 with an average of 0.48. For the assignment of these markers to BAC clones, a PCR-based strategy was established to screen the “Morex”-BAC library. By using this strategy BAC addresses were obtained for a total of 127 mapped EST-SSRs, which may provide at least two markers located on a single BAC. This observation is indicative of an uneven distribution of genes and may lead to the identification of gene-rich regions in the barley genome.