Assembly of the barley (Hordeum vulgare L.) genome requires high resolution maps for aligning contig-based physical maps along chromosomes. Genetic maps lack accurate information on the physical position of almost half of the barley genome located in recombination-poor regions. Radiation hybrid (RH) mapping is an alternative approach, which is based on radiation-induced chromosomal deletions. In this study, an RH population for barley chromosome 3H was developed. Genotyping 373 3H-RH lines with 113 markers resulted in an RH map with an average resolution of 2.22 Kb. Compared to an analogous genetic map, the 3H-RH map resolution was 9.53-X higher, reaching to >262.40-X better resolution in the centromeric region. We suggest that RH maps would facilitate assembly of the barley genome. For future RH studies of the barley genome, an optimum genotyping platform, consisting of 400,536 barley-specific repeat junction markers (RJMs), was developed.