Active females demonstrate increased risk for musculoskeletal injuries relative to equivalently-trained males. Although gender differences in factors such as passive laxity, skeletal geometry and kinematics have been examined, the effect of gender on active muscle stiffness has not been reported. Stiffness of the active quadriceps and hamstrings musculature were recorded during isometric knee flexion and extension exertions from twelve male and eleven female subjects. A second-order biomechanical model of joint dynamics was used to quantify stiffness from the transient motion response to an angular perturbation of the lower-leg. Female subjects demonstrated reduced active stiffness relative to male subjects at all torque levels, with levels 56-73% of the males. Effective stiffness increased linearly with the torque load, with stiffness increasing at a rate of 3.3 Nm/rad per unit of knee moment in knee flexion exertions (hamstrings) and 6.6 Nm/rad per unit of knee moment extension exertions (quadriceps). To account for gender differences in applied moment associated with leg mass, regressions analyses were completed that demonstrated a gender difference in the slope of stiffness-versus-knee moment relation. Further research is necessary to identify the cause of the observed biomechanical difference and implications for controlling injury.