This study evaluated seven different frames of reference used for tibial component rotation in total knee arthroplasty (TKA) to determine which ones showed good reliability between bone specimens. An optoelectronic system based around a computer-assisted surgical navigation system was used to measure and locate 34 individual anatomical landmarks on 40 tibias. Each particular frame of reference was reconstructed from a group of data points taken from the surface of each bone. The transverse axis was used as the baseline to which the other axes were compared, and the differences in angular rotation between the other six reference frames and the transverse axis were calculated. There was high variability in the tibial rotational alignment associated with all frames of reference. Of the references widely used in current TKA procedures, the tibial tuberosity axis and the anterior condylar axis had lower standard deviations (6.1° and 7.3°, respectively) than the transmalleolar axis and the posterior condylar axis (9.3° for both). In conclusion, we found high variability in the frames of reference used for tibial rotation alignment. However, the anterior condylar axis and transverse axis may warrant further tests with the use of navigation. Combining different frames of reference such as the tibial tuberosity axis, anterior condylar axis and transverse axis may reduce the range of errors found in all of these measurements.