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Histological and chemical characteristics of longissimus muscle and physical measurements of scapular and vertebral cartilage and bone were related to the tenderness of the beef longissimus. The 195 wholesale ribs were selected from carcasses differing widely in physiological maturity. Smaller diameter muscle fibers, longer sarcomeres, shorter muscle fiber fragments following homogenization, lower percentages of wavy fibers, decreased collagen content, increased percentages of soluble collagen, lower myofibril fragmentation scores, increased percentages of fat, decreased percentages of moisture and smaller areas of expressible juice were associated (P < 0.05 or P < 0.01) with increases in tenderness of the beef longissimus. Among physiological maturity indicators, muscle fiber diameter, total pigment concentration and hardness of scapular cartilage and vertebral bone were most closely related to the variability in tenderness of the beef longissimus and were associated with advancements in physiological maturity. Although most of the relationships in this study were of small magnitude, these data generally support the utilization of fatness‐maturity interactions across very wide ranges in physiological maturity for the stratification of beef carcasses according to tenderness.