The purpose of this study was to compare the recruitment patterns in hamstring muscles during hip extension exercise by electromyography (EMG) and muscle functional magnetic resonance imaging (mfMRI). Six male volunteers performed 5 sets of 10 repetitions of the hip extension exercise. Electromyography (EMG) activity during the exercise was recorded for the biceps femoris long head (BFlh), semitendinosus (ST), and semimembranosus (SM) muscles; mfMRI T2 values and cross-sectional areas (CSAs) of the same muscles were measured at rest, immediately after, 2 and 7 days after the exercise. The study found that EMG of the BFlh and SM were significantly higher than that of the ST. Immediately after the exercise, the T2 value and CSA changes in the SM showed a significant increase. It was concluded that the BFlh and SM were selectively recruited during the hip extension exercise.