In this study, chemical composition, fatty acids, collagen solubility, calpains, and troponin T degradation were investigated in various muscles of native Thai cattle (NTC). Five muscles, namely Bicep femoris, Infraspinatus, Longissimus thoracis, Supraspinatus and Semimembranosus were sampled from five Kho Isaan bulls. Moisture content of Longissimus thoracis was lower (p < 0.05) than others. Protein content of Longissimus thoracis, Bicep femoris and Semimembranosus was higher (p < 0.05) than that of Infraspinatus and Supraspinatus. Infraspinatus and Longissimus thoracis had higher fat content (p < 0.05) than Bicep femoris and Semimembranosus. Muscle pH, color, and drip loss did not differ between muscles. Semimembranosus and Supraspinatus showed higher cook loss (p < 0.05) than Infraspinatus and Longissimus thoracis. Infraspinatus had the lowest Warner–Bratzler shear force (p < 0.05). Supraspinatus, Infraspinatus and Bicep femoris had higher (p < 0.05) soluble, insoluble and total collagen content than Semimembranosus and Longissimus thoracis. Infraspinatus had higher collagen solubility than Semimembranosus and Supraspinatus. Collagen content and solubility were not affected by ageing time. The relative band intensities of intact troponin T and its degradation product at 2- and 14-days postmortem were higher in Longissimus thoracis than in other muscles. Infraspinatus and Supraspinatus had significantly higher expression of m-calpain and calpastatin genes than Longissimus thoracis, Bicep femoris, and Semimembranosus. Total fatty acid was higher while the percentage of C22:6n-3 was lower in Infraspinatus and Longissimus thoracis (p < 0.05) than in other muscles. These results show differences in specific nutritional, technological and eating quality characteristics of NTC muscles and thus provide information for optimizing storage and processing conditions for various NTC muscles.