AbstractThe structural and functional differences of individual hamstrings have not been sufficiently evaluated. This study aimed to clarify the morphological architecture of the hamstrings including the superficial tendons in detail using isolated muscle specimens, together with quantification of structural parameters of the muscle. Sixteen lower limbs of human cadavers were used in this study. The semimembranosus (SM), semitendinosus (ST), biceps femoris long head (BFlh), and biceps femoris short head (BFsh) were dissected from cadavers to prepare isolated muscle specimens. Structural parameters, including muscle volume, muscle length, fiber length, sarcomere length, pennation angle, and physiological cross‐sectional area (PCSA) were measured. In addition, the proximal and distal attachment areas of the muscle fibers were measured, and the proximal/distal area ratio was calculated. The SM, ST, and BFlh were spindle‐shaped with the superficial origin and insertion tendons on the muscle surface, and the BFsh was quadrate with direct attachment to the skeleton and BFlh tendon. The muscle architecture was pennate in the four muscles. The four hamstrings possessed either of two types of structural parameters, one with shorter fiber length and larger PCSA, as in the SM and BFlh, and the other with longer fiber length and smaller PCSA, as in the ST and BFsh. Sarcomere length was unique in each of the four hamstrings, and thus the fiber length was suitably normalized using the average sarcomere length for each, instead of uniform length of 2.7 μm. The proximal/distal area ratio was even in the SM, large in the ST, and small in the BFsh and BFlh. This study clarified that the superficial origin and insertion tendons are critical determinants of the unique internal structure and structural parameters representing the functional properties of the hamstring muscles.