Female athletes are more likely to sustain an anterior cruciate ligament (ACL) injury. Knee laxity, hyperextension and limb dominance have been suggested as possible factors contributing to the knee injury. The aims of this study were to investigate the physiological anterior knee laxity between the dominant and non-dominant limb and in healthy young females with and without hyperextension knees. Forty-two healthy young females, 21 with hyperextension knees, were recruited voluntarily for this study. The subjects were tested with KT-2000 knee ligament arthrometer at both knees with flexion 30° to obtain the anterior tibial displacements at loadings of 45, 67, 89 and 134 N. The initial and terminal stiffnesses were further calculated and analyzed to demonstrate the differences in the characteristics of knee laxity between limbs and groups. The results showed that there was no significant displacement difference between hyperextension and non-hyperextension groups. However, different physiological anterior laxities were illustrated for the different limbs and groups. The non-dominant side of the hyperextension group had significantly smaller terminal stiffness than that of the non-hyperextension group. The dominant side of the hyperextension group had larger laxity than the non-dominant side in the higher loading conditions. These findings may explain hyperextension knees are at greater risk of sustaining an ACL injury.