Abstract: Calpastatin plays an important role in muscle growth and meat quality formation. To investigate the molecular basis of the meat quality of hybrid F₁ from Ctenopharyngodon idellus (♀) × Squaliobarbus curriculus (♂), the full-length cDNAs of CiCAST, ScCAST and their hybrid F₁ CAST were cloned by using RACE technology, and structure differences were analyzed by bioinformatics tools in this study. The results showed that the full-length cDNA of CiCAST, ScCAST and hybrid F₁ CAST were 3036, 3165 and 3086 bp in length and encoded 901, 893 and 904 amino acids, respectively; the predicted molecular weights were 93.72, 92.77 and 94.02 kDa, and the theoretical isoelectric points were 5.92, 6.01 and 6.02, respectively. The nucleotide sequence similarity of CAST between F₁ CAST and CiCAST was 94.52% and was 90% between F₁ CAST and ScCAST. The three CAST proteins contained four typical calpain inhibitory domains, which had typical heptapeptide structures. A prediction of the phosphorylation sites showed that there were 73, 82 and 75 potential phosphorylation modification sites in the amino acid residues of CiCAST, ScCAST and F₁CAST, respectively. The CAST tertiary structure analysis showed that CiCAST, ScCAST and F₁CAST contained 24, 12 and 20 β-folds, which were all formed into β-chain structures. It was concluded that F₁ CAST was more similar to CiCAST in sequence similarity, number of phosphorylation sites, protein structure and evolutionary status. The results provide a molecular basis for the elucidation of meat quality formation in hybrid F1 fish.