Surgical access to the posterior knee poses a high-risk for neurovascular damage. The study aimed to define the popliteal fossa by reliable bony landmarks and comprehensively mapping the neurovascular structures for application in posterior knee surgery. Forty-five (20 male, 25 female) embalmed adult cadaveric knees were included. The position of the small saphenous vein (SSV), medial cutaneous sural nerve (MCSN) and lateral cutaneous sural nerv (LCSN), tibial nerve (TN) and common fibular nerve (CFN) nerves, and popliteal vein (PV) and popliteal artery (PA) were determined in relation to either medial (MFE) or lateral (LFE) femoral epicondyles, medial (MTC) and lateral (LTC) tibial condyles and the midpoint between the MFE and MTC and LFEF and LTC. The distance between the MFE and the PA, PV, TN, MCSN, and SSV was 38.4±12.1 mm, 38.4±12.9 mm, 39.4±10.2 mm, 39.2±14.0 mm and 37.6±12.5 mm respectively for males and 34.6±4.9 mm, 32.8±5.6 mm and 38.0±8.1 mm 38.8±10.1 mm and 37.9±8.2 mm respectively for females. The distance between LFE and the CFN and LCSN was 13.4±8.2 mm and 24.9±7.3 mm respectively for males and 8.4±9.1 mm and 18.4±10.4 mm respectively in females. This study defined the popliteal fossa by reliable bony landmarks and provided a comprehensive map of the neurovascular structures and will help to avoid injuries to the important neurovascular structures.