There are two twist genes in vertebrates, namely twist1 and twist2, which have a regulatory effect on bone development. To explore the relationship between twist1, 2 and intermuscular bone development in barbel steed (Hemibarbus labeo), cDNAs of the twist1 and twist2 genes of H. labeo were cloned and sequenced. Sequence analysis revealed that H. labeo TWIST1 and TWIST2 were conserved in most of the teleosts, and they all had a helix-loop-helix (HLH) domain and a tryptophan-arginine (WR) domain. Phylogenetic tree analysis indicated that H. labeo twist1 and twist2 were closely related to that of zebrafish (Danio rerio). Expression of twist1 and twist2 transcripts were detected in all the tested tissues. twist1 was the highestly expressed in the brain, followed by the heart, and the lowest in the spleen. twist2 had higher expression levels in skin and gill, and the lowest expression in spleen. The transcript levels of the twist1 and twist2 gene significantly changed during the four developmental stages of intermuscular bone development, suggesting that there is a certain correlation between twist genes and intermuscular bone development. This study, for the first time, cloned the twist genes in economic fish and revealed the correlation between twist genes and intermuscular bone development. The results will provide basic data for further investigation of twist gene function and its regulation role in intermuscular bone development.