New high-molecular-weight glutenin (HMW glutenin) sequences isolated from six Psathyrostachys juncea accessions by thermal asymmetric interlaced PCR differ from previous sequences from this species. They showed novel modifications in all of the structural domains, with unique C-terminal residues, and their N-terminal lengths were the longest among the HMW glutenins reported to date. In their repetitive domains, there were three repeatable motif units: 13-residue [GYWH(/I/Y)YT(/Q)S(/T)VTSPQQ], hexapeptide (PGQGQQ), and tetrapeptide (ITVS). The 13-residue repeats were restricted to the current sequences, while the tetrapeptides were only shared by D-hordein and the current sequences. However, these sequences were not expressed as normal HMW glutenin proteins because an in-frame stop codon located in the C-termini interrupted the intact open reading frames. A phylogenetic analysis supported different origins of the P. juncea HMW glutenin sequences than that revealed by a previous study. The current sequences showed a close relationship with D-hordein but appeared to be more primitive.