Background: Barley prolamin is a protein affecting end-use quality, malt production, and feeding value. Barley B-hordeins account for 70-80% of total prolamins and have significant effects on barley quality. It is of great importance to study genes encoding B-hordeins. Methods: Eighteen different barley varieties were used to identify new B-hordein genes. Genomic DNA was extracted from barley seedlings using a modified CTAB procedure, and PCRs were performed to amplify B-hordein genes using newly designed primers based on the previously published B-hordein gene sequences (Accessions X03103 and X87232). The B-hordein genes registered in GenBank were also obtained from NCBI. Gene sequences were analyzed using the software DNAman. Phylogenetic analysis was conducted by the method of Maximum Likelihood. Results: A total of 20 complete B-hordein gene sequences were obtained, including 14 putative active genes and 6 pseudogenes. Sequence variations were identified in all 20 genes compared with 72 B-hordein genes from GenBank (including 27 genes we previously identified). The deduced amino-acid sequences of 13 genes had a typical set of 8 conserved cysteine residues, and the other one had 7 cysteines and this gene may have positive effects on barley quality. Sequence analysis and phylogenetic analysis indicated obvious differences between the three kinds of B-hordeins ended with an amino acid V, H or Y. Conclusion: 20 new B-hordein gene sequences were identified, including one with 7 cysteine residues. 92 B-hordein genes were classified into three types (V-type, H-type and Y-type), of which the V-type should be the dominant, original B-hordein genes, while the H-type and the Y-type might be the variants of the V-type. The results provided a reference for further characterizing the B-hordein genes and for breeding quality barley varieties.