Objective: The objectives of this study were to explore the expression patterns of myosin heavy chain (<i>MyHC</i>) genes of different skeletal muscles from Chinese cattle, and to investigate the relationship between myofiber characteristics and meat quality of <i>M. longissimus lumborum</i> (LL), <i>M. psoas major</i> (PM), and <i>M. semimembranosus</i> (SM) from Chinese Luxi and Qinchuan cattle.Methods: Three major muscles including LL, PM, and SM from Chinese Luxi cattle and Chinese Qinchuan cattle were used in this study. The myofiber characteristics were measured by histochemical analysis. The MyHC isoforms expression was evaluated by real-time quantitative polymerase chain reaction. Quality traits including pH value, meat color, cooking loss, Warner-Bratzler shear force (WBSF) and sarcomere length were determined at day 5 postmortem.Results: PM muscle had higher pH value, <i>a</i>* value, sarcomere length and lower WBSF value compared to LL and SM muscles (p<0.05). Numbers of type I myofiber and the relative expression of <i>MyHC I</i> mRNA in PM muscle were higher than those of LL and SM muscles (p<0.05). Myofiber diameter of PM muscle was lower than that of LL and SM muscles, regardless of myofiber types (p<0.05).Conclusion: According to the stepwise linear regression analyses, tenderness was influenced by myofiber characteristics in all three examined muscles. Tenderness of beef muscles from Qinchuan and Luxi cattle could be improved by increasing numbers of type I myofiber.