Background: Both the range of motion (ROM) technique and the tibial tubercle landmark (TTL) technique are frequently used to align the tibial component into proper rotational position during total knee arthroplasty (TKA). The aim of the study was to assess the intra-operative differences in tibial rotation position during computer-navigated primary TKA using either the TTL or ROM techniques. The ROM technique was hypothesized to be a repeatable method and to produce different tibial rotation positions compared to the TTL technique. Methods: A prospective, observational study was performed to evaluate the antero-posterior axis of the cut proximal tibia using both the ROM and the TTL technique during primary TKA without postoperative clinical assessment. Computer navigation was used to measure this difference in 20 consecutive knees of 20 patients who underwent a posterior stabilized total knee arthroplasty with a fixed-bearing polyethylene insert and a patella resurfacing. Results: The ROM technique is a repeatable method with an interclass correlation coefficient (ICC2) of 0.84 (p < 0.001). The trial tibial baseplate was on average 4.56 degrees externally rotated compared to the tubercle landmark. This difference was statistically significant (p = 0.028). The amount of maximum intra-operative flexion and the pre-operative mechanical axis were positively correlated with the magnitude of difference between the two methods. Conclusions: It is important for the orthopaedic surgeon to realise that there is a significant difference between the TTL technique and ROM technique when positioning the tibial component in a rotational position. This difference is correlated with high maximum flexion and mechanical axis deviations.