Brachypodium distachyon, a small wild grass within the Pooideae family, is a new model organism for exploring the functional genomics of cereal crops. It was shown to have close relationships to wheat, barley and rice. Here, we describe the molecular characterisation and evolutionary relationships of high molecular weight glutenin subunits (HMW-GS) genes from B. distachyon. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), high performance capillary electrophoresis (HPCE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses demonstrated that there was no HMW-GS expression in the Brachypodium grains due to the silencing of their encoding genes. Through allele-specific polymerase chain reaction (AS-PCR) amplification and cloning, a total of 13 HMW-GS encoding genes from diploid, tetraploid and hexaploid Brachypodium species were obtained, and all of them had typical structural features of y-type HMW-GS genes from common wheat and related species, particularly more similar to the 1Dy12 gene. However, the presence of an in-frame premature stop codon (TAG) at position 1521 in the coding region resulted in the conversion of all the genes to pseudogenes. Further, quantitative real-time PCR (qRT-PCR) analysis revealed that HMW-GS genes in B. distachyon displayed a similar trend, but with a low transcriptional expression profile during grain development due to the occurrence of the stop codon. Phylogenetic analysis showed that the highly conserved Glu-1-2 loci were presented in B. distachyon, which displayed close phylogenetic evolutionary relationships with Triticum and related species.