Crop wild relatives (CWR) provide an important source of allelic diversity for any given crop plant species for counteracting the erosion of genetic diversity caused by domestication and elite breeding bottlenecks. Hordeum bulbosum L. is representing the secondary gene pool of the genus Hordeum. It has been used as a source of genetic introgressions for improving elite barley germplasm (Hordeum vulgare L.). However, genetic introgressions from H. bulbosum have yet not been broadly applied, due to a lack of suitable molecular tools for locating, characterizing, and decreasing by recombination and marker-assisted backcrossing the size of introgressed segments. We applied next-generation sequencing (NGS) based strategies for unlocking genetic diversity of three diploid introgression lines of cultivated barley containing chromosomal segments of its close relative H. bulbosum. Firstly, exome capture-based (re)-sequencing revealed large numbers of single nucleotide polymorphisms (SNPs) enabling the precise allocation of H. bulbosum introgressions. This SNP resource was further exploited by designing a custom multiplex SNP genotyping assay. Secondly, two-enzyme-based genotyping-by-sequencing (GBS) was employed to allocate the introgressed H. bulbosum segments and to genotype a mapping population. Both methods provided fast and reliable detection and mapping of the introgressed segments and enabled the identification of recombinant plants. Thus, the utilization of H. bulbosum as a resource of natural genetic diversity in barley crop improvement will be greatly facilitated by these tools in the future.