The purpose of this study was to examine whether muscle architecture of the long head of biceps femoris (BF) and semitendinosus (ST) muscles varies along their length. The ST and BF muscles were dissected and removed from their origins in eight cadaveric specimens (age range 67.8-73.4 years). One-way analysis of variance designs were used to compare fascicle length (FL), pennation angle (PA) and muscle thickness (MT) between proximal, mid-belly and distal positions. Tendon and muscle length properties were also quantified. For the BF muscle, one-way analysis of variance tests showed a higher PA (23.96±3.82°) and FL (7.12±0.48 cm) proximally than distal positions (PA=17.78±1.95° and FL=6.35±0.89 cm, respectively). For the ST, there was a significantly (p<0.05) lower PA (8.81±1.22°) and FL (13.10±1.54 cm) proximally than distally (PA=14.69±1.09° and FL=15.49±2.30 cm, respectively). Muscle thickness significantly increased from distal to more proximal positions (p<0.05). These data suggest that the ST and BF architecture is not uniform and that measurement of these parameters largely depends on the measurement site. Modeling these muscles by assuming a uniform architecture along muscle length may yield less accurate representation of human hamstring muscle function.