Mouse models are increasingly used in current research on motor disorders. In mice, the myoelectrical activation of thigh muscles during locomotion has not yet, however, been investigated in depth. Especially intramuscular coordination has hardly been clarified. Therefore, the aims of this study were to characterize myoelectrical activity in the vastus lateralis (VL) and the biceps femoris (BF) muscle of the healthy mouse for reference purposes. The VL and the BF muscles of 12 healthy mice performing a total of 1985 steps during treadmill locomotion were investigated with two subcutaneous arrays each incorporating four electrodes. Eight-channel EMG was recorded simultaneously with high-speed videography. The EMG curves of each step were rectified and smoothed by calculating root mean square (RMS) profiles and then time-normalized for comparisons within and between animals. The EMG-activity of both muscles increased during late swing phase. The VL activity rose steeply and peaked during mid-stance phase, while the biceps activity reached a plateau during early stance phase. With increasing gait velocity, stance time decreased. The increase in gait velocity was also associated with greater EMG amplitudes. The results suggest that the BF lifts the lower hind leg during swing phase and stabilizes the leg during stance, while the VL bears the weight of the body during the stance phase.