The B-hordein gene family was analyzed from two Tibetan hull-less barley cultivars Z09 and Z26 (Hordeum vulgare subsp. vulgare). Fourteen B-hordein genes, designated BZ09-2 to BZ09-5 (from Z09) and BZ26-1 to BZ26-10 (from Z26), were sequenced. Seven of them, similar to a previously reported BZ09-1 from Z09, were predicted to encode putative active proteins each with a signal peptide, a repetitive domain, and a C-terminal region; seven of them were predicted to be pseudogenes. The B-hordein gene family was analyzed using all known representatives of B-hordein sequences and two most similar LMW-GSs of Triticum aestivum. Alignment of these seven putative proteins with known B-hordeins and two most similar LMW-GSs of T. aestivum revealed that they shared a common motif. A large variation was observed between numbers of repeat units of predicted B-hordeins of Z26 and Z09. Phylogenetic analysis revealed that all BZ26 clones were clustered in a subgroup, and BZ09-1 formed another subgroup by itself in the putative eight active genes. In addition, six 5'-upstream regulatory sequences of the B-hordein genes were isolated from the two accessions by a single oligonucleotide nested PCR, and several different mutations were identified in the cis-acting element GLM and two distinctive sequences (Z09P-2 and Z26P-3). Phylogenetic analysis of 5'-upstream regulatory regions of the B-hordein genes showed that members from the same accession were clustered together except for two distinct members. Quantitative real time PCR analysis indicated distinct expression levels of B-hordein genes in four developing stages of developing grains in two accessions. These findings suggest B-hordein genes have intrinsic differences between accessions, and this knowledge will be useful for incorporating the B-hordeins protein in barley breeding programs.