The aim of present study was to compare electromyography activity of selected leg-dominant lower limb muscles during stance phase of running on treadmill and overground. Fourteen male students ran at 3.3 m/s in both treadmill and overground conditions. Electromyography activity of some selected lower limb muscle was recorded during initial 50% and terminal 50% of stance phase. Paired t-test was employed for data analyses. The results showed a significant difference in total activity of selected lower limb muscles between treadmill and overground running conditions (P<0.05). Rectus femoris, vastus medialis, vastus lateralis, and biceps femoris activation during overground running were found significantly higher than running on the treadmill in initial 50% stance phase (P<0.05). No significant electromyography change was observed for selected muscles during terminal 50% of stance phase in both treadmill and overground conditions (P>0.05). It was concluded that treadmills running condition may be possibly useful in designing specific training programs that are aimed to control or reduce lower extremity muscles activity. According to the results of this study, treadmills running condition caused lower muscle activity consequently, may increase biomechanical efficiency or used in clinical setting.