Crop wild relatives have been recognized as a source of beneficial traits to a given crop species and to overcome the erosion of genetic diversity resulting from domestication and breeding programs. Hordeum bulbosum L. is the only representative of the secondary gene pool of barley (Hordeum vulgare L.) and it has been found as a valuable source of genetic diversity for barley improvement. Since the 1990s a considerable number of barley/H. bulbosum introgression lines (ILs) have been generated. These ILs harbor a diverse set of desirable traits. So far, the efficient utilization of such ILs for improving elite barley germplasm has been hampered, largely due to the lack of suitable molecular tools for locating introgressed segments. Thus, the focus of the present study was to establish suitable and state-of-the-art molecular tools and resources for the efficient utilization of H. bulbosum traits in barley breeding and research.