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This study aims to determine optimal locations on the lower limbs for skin-mounted markers representing the tibial segment in three-dimensional (3D) gait analysis. It was predicted that markers located on the anterior tibial crest and malleoli would be least susceptible to soft tissue movement. Ten retro-reflective markers were attached to each tibial segment for 20 participants. Participants performed 10 walking trials and two different range-of-movement tasks (knee flexion/extension and ankle plantarflexion/dorsiflexion). The results showed a subset of four markers with inter-marker pair distances on the tibia have less than 1.6mm variation (standard deviation (S.D.)) during walking. Minimal variation was also found in isolated ROM tasks, where marker pairs showed variability of less than 2.2mm. Other marker locations, the femoral epicondyles and the tibial tuberosity varied up to 4mm during walking and up to 11mm …