In adult bovine skeletal muscle, it expressed four isoforms of Myosin heavy chain (MyHC) gene, MyHC-I, MyHC-IIa, MyHC-IIb, and MyHC-IIx that are translated into different structural protein myofibrils, and then further form different types of muscle fiber. In the studies, our objective is to reveal the expression patterns of MyHC genes in longissimus dorsi (Ld), semitendinosus (Se) and soleus (Sol) of Simmental hybrids cattle, and their association with intramuscular fat (IMF) content and meat shearing force (MSF). The muscle tissue of Ld, Se and Sol were collected from 6, 12 and 36-month old Simmental hybrids respectively, then the expression levels of MyHCs were examined by real-time PCR, at the same time, IMF, MSF and muscle type were measured with chemical assessment, shearing force measurer and immunostaining respectively. Our results showed that t Ld, Se, and Sol expressed MyHC-I, MyHC-IIa and MyHC-IIx isoforms but not MyHC-IIb, furthermore MyHC-I, MyHC-IIa and MyHC-IIx had different expression patterns in different skeletal muscle. The expression of MyHC-I in Se and Sol, MyHC-IIa in Ld, Se, and Sol, and MyHC-IIx in Sol was decreased with increasing age. The highest expression of MyHC-I in Ld, and MyHC-IIx in Ld and Se was observed in 12-month-old animals. The percentage of type-IIa fiber approximately occupied 70-80 % among various muscle fiber of Ld, Se and Sol. The percentage of different type fiber was not related to IMF content and MSF, but the expression levels of MyHC-I and MyHC-IIa were negatively related to IMF content (r = -0.724, and -0.681, respectively) and MSF (r = -0.672, and -0.641, respectively). The expression level of MyHC-IIx was also negatively related to MSF (r = -0.655). In conclusion, MyHC gene might be considered as a negative factor in genetic selection of IMF content and MSF.