Cultivated barley (Hordeum vulgare L.) is well known as one of the most widely cultivated crops in the world and as an extensively studied plant species in the field of genetics. In recent years, despite its very large genome size (ca. 5,000 Mb), the research resources needed for barley genomic studies have become available, including a large number of expressed sequence tags (ESTs). These have been widely used for barley genome analyses, such as DNA marker-generation and the construction of microarrays. However, the availability of a large-insert genomic library, which is also essential for genomic studies, has been relatively limited in the barley research community. We described here the construction and characterization of a barley bacterial artificial chromosome (BAC) library, using the Japanese malting barley variety ‘Haruna Nijo’. The BAC library consisted of 294,912 clones arrayed in 768 384-well microtiter plates. The average size of each cloned insert was estimated to be 115.2 kb, with approximately 0.5% of the clones lacking inserts. Chroloplast DNAs were present in about 1.7% of the library. Thus, the genomic coverage of the ‘Haruna Nijo’ BAC library was estimated to be about 6.6 genome-equivalents. In order to rapidly identify specific BAC clones, we developed a screening strategy that combined PCR analysis of pooled BAC DNAs with colony hybridization. Using this screening scheme, we investigated the genomic coverage of this BAC library, using 13 locus-specific ESTs and a sequence-tagged site marker. By screening the whole library with individual markers, we identified an average of 5.1 clones per marker. This screening scheme also enabled us to rapidly construct a physical contig spanning a region of approx. 190 kb around the HvBRI1 locus, where the mutation responsible for the semi-dwarf plant type ‘uzu’ is located. These results indicate that the ‘Haruna Nijo’ BAC library will be useful for barley genomic studies.