A high-resolution transcript linkage map of barley was created using a single doubled haploid (DH) mapping population, only 3'-end expressed sequence tags (ESTs) and only PCR-based assays. Cultivar 'Haruna Nijo' and an ancestral wild-form accession 'H602' were used as EST donors and crossing parents of the mapping population. Of the 10,366 primer sets developed from a non-redundant set of 3'EST sequences, 7700 sets generated useful amplicons and 3975 (52%) showed polymorphisms between the mapping parents. Of these, 2890 (28% of the total) were mapped by single nucleotide polymorphisms (1717), cleaved amplified polymorphic sequence (933) and INDELs (240). The present work involves an estimated 9% of the genes of barley. Of the mapped ESTs, 2689 (93%) are formatted in the Affymetrix Barley 1 GeneChip and full-length cDNA sequences are available for 1039 (36%). Mapped ESTs show highest similarity with sequences in the wheat gene index (93%) and moderate similarity with rice (50%). Comparison of mapped EST positions and the rice pseudomolecule indicated collinear regions between two species; these are particularly conserved for the entire barley chromosome 3H and rice chromosome 1. These mapped genes, together with a systematically developed set of genetic resources, will make it easier to directly clone genes showing simple inheritance and to determine the genetic basis of complex traits. The information will contribute to the development of a framework for the physical mapping of barley, which is necessary for genome sequencing.