As an important plateau cereal crop, hulless barley is the principal food for the Tibetan people in China. ADP-glucose pyrophosphorylase (AGPase) is considered as the key enzyme for starch biosynthesis in plants. In this study, cDNAs encoding the small subunit (SSU I) and large subunit (LSU I) of AGPase were isolated from hulless barley. The results showed that SSU I and LSU I were 1438 and 1786 bp in length with a complete open reading frame (ORF) of 1419 and 1572 bp. The ORF-encoded polypeptides of 472 and 523 amino acids were having calculated molecular masses of 52.01 and 58.23 kDa, and the pI values were 5.59 and 6.30. In addition, phylogenetic analysis showed that SSU I and LSU I had the same phylogenetic trends with some species. Furthermore, expression levels in different growth periods and tissues of two hulless barley varieties were analyzed by quantitative reverse transcription-polymerase chain reaction. Gene expression levels of SSU I and LSU I were consistent with the total starch accumulation rate in endosperm. In conclusion, our data confirmed that SSU I and LSU I played an important role in hulless barley starch synthesis.