Wheat-barley chromosome addition lines are useful genetic resources for a variety of studies. In this study, transcript accumulation patterns in Betzes barley, Chinese Spring wheat, and Chinese Spring-Betzes chromosome addition lines were examined with the Barley1 Affymetrix GeneChip probe array. Of the 4014 transcripts detected in Betzes but not in Chinese Spring, 365, 271, 265, 323, 194, and 369 were detected in wheat-barley disomic chromosome addition lines 2(2H), 3(3H), 4(4H), 7(5H), 6(6H), and 1(7H), respectively. Thus, 1787 barley transcripts were detected in a wheat genetic background and, by virtue of the addition line in which they were detected, were physically mapped to barley chromosomes. We validated and extended our approach to physically map barley genes to the long and short arms of chromosome 6(6H). Our physical map data exhibited a high level of synteny with homologous sequences on the wheat and/or rice syntenous chromosomes, indicating that our barley physical maps are robust. Our results show that barley transcript detection in wheat-barley chromosome addition lines is an efficient approach for large-scale physical mapping of genes.