The objective of this study is to compare the muscular activity of lower extremity muscles while running on treadmill and on overground surfaces. A total of 13 experienced heel-to-toe runners participated in the study. Electromyographic (EMG) data of four lower extremity muscles, including rectus femoris, tibialis anterior, biceps femoris, and gastrocnemius, were collected using the Noraxon EMG system while running on a treadmill and on overground surfaces at a running speed of 3.8 m/s. The obtained data were then analyzed. In this study, throughout the stance phase, the EMG values in the rectus femoris (P<0.01) and the biceps femoris (P<0.05) were higher while running on overground surfaces than those on a treadmill. The EMG values in the rectus femoris (P<0.05) and the biceps femoris (P<0.05) were also higher on concrete than those on grass in the stance phase. Results showed that the muscle activity was significantly different in treadmill running than in overground running. The difference in muscle activity while running on different overground surfaces was also found in this study. Kinematic adjustment of the lower extremity may explain the EMG difference while running on different surfaces.