Rotational stability of the knee has been traditionally difficult to quantify, limiting the ability of the orthopedic community to determine the potential role of rotational laxity in the etiology of anterior cruciate ligament (ACL) injuries. The purposes of this multicenter cohort study were to evaluate the reliability of a robotic axial rotation measurement system, determine whether the uninjured knees of patients that had previous contralateral ACL reconstruction demonstrated different rotational biomechanical characteristics than a group of healthy volunteers, and determine whether knee rotational biomechanical characteristics differ between male and female non-injured limbs in groups of both healthy volunteers and patients with a previous contralateral ACL injury. Fourteen healthy volunteers and 79 patients with previous unilateral ACL injury participated in this study. Patients were tested using a computerized tibial axial rotation system. Only the normal (non-operated) knee data were used for analysis. In order to assess the reliability of the robotic measurement system, 10 healthy volunteers were tested daily over four consecutive days by four different examiners. Rotational laxity and compliance measures demonstrated excellent reliability (ICC = 0.97). Patients with a contralateral ACL injury demonstrated significantly increased tibial internal rotation (20.6° vs. 11.4°, P < 0.001) and reduced external rotation (16.7° vs. 26.6°, P < 0.001) compared to healthy volunteers. Females demonstrated significantly increased internal and external rotation, as well as significantly increased rotational compliance compared with males (P < 0.05). Computer-assisted measurement techniques may offer clinicians an accurate, reliable, non-invasive method to select the most appropriate preventative or surgical interventions for patients with increased knee rotational laxity.