Gait analysis of trans-tibial (TT) amputees discloses asymmetries in gait parameters between the amputated and sound legs. The present study aimed at outlining differences between both legs with regard to kinematic parameters and activity of the muscles controlling the knees. The gait of 14 traumatic TT amputees, walking at a mean speed of 74.96 m/min, was analysed by means of an electronic walkway, video camera, and portable electromyography system. Results showed differences in kinematic parameters. Step length, step time and swing time were significantly longer, while stance time and single support time were significantly shorter on the amputated side. A significant difference was also found between knee angle in both legs at heel strike. The biceps femoris/vastus medialis ratio in the amputated leg, during the first half of stance phase, was significantly higher when compared to the same muscle ratio in the sound leg. This difference was due to the higher activity of the biceps femoris, almost four times higher than the vastus medialis in the amputated leg. The observed differences in time-distance parameters are due to stiffness of the prosthesis ankle (the SACH foot) that impedes the normal forward advance of the amputated leg during the first half of stance. The higher knee flexion at heel strike is due to the necessary socket alignment. Unlike in the sound leg, the biceps femoris in the amputated leg reaches maximal activity during the first half of stance, co-contracting with the vastus medialis, to support body weight on the amputated leg. The obtained data can serve as a future reference for evaluating the influence of new prosthetic components on the quality of TT amputee's gait.