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Objective To investigate the anatomical characteristics of the biceps femoris short head (BS) and determine the optimal needle placement for BS examination. Methods Twenty-one lower limbs were dissected. The distances from the medial and lateral margins of the biceps femoris long head (BL) tendon to the common fibular nerve (CFN) (M_CFN_VD and L_CFN_VD, respectively) and the distance from the lateral margin of the BL tendon to the lateral margin of the BS (L_BS_HD) were measured 5 cm proximal to the tip of the fibular head (P1), four fingerbreadths proximal to the tip of the fibular head (P2), and at the upper apex of the popliteal fossa (P3). Results The BS was located lateral to the BL tendon. The CFN was located along the medial margin of the BL tendon. The median values were 2.0 (P1), 3.0 (P2), and 0 mm (P3) for M_CFN_VD; and 17.4 (P1), 20.2 (P2), and 21.8 mm (P3) for L_CFN_VD; and 8.1 (P1), 8.8 (P2), and 13.0 mm (P3) for L_BS_VD. Conclusion The lateral approach to the BL tendon was safer than the medial approach for examining the BS. Amore proximal insertion site around the upper apex of the popliteal fossa was more accurate than the distal insertion site. In this study, we propose a safer and more accurate approach for electromyography of the BS.