Background Understanding hamstring muscle activation patterns in resistance training exercises may have implications for the design of strength training and injury prevention programs. Unfortunately, surface electromyography studies have reported conflicting results regarding hamstring muscle activation patterns in women. Objectives To determine the spatial patterns of hamstring muscle activity during the 45° hip extension and Nordic hamstring exercises in women using functional magnetic resonance imaging (fMRI). Methods This was a cross-sectional study in which 6 recreationally active women with no history of lower-limb injury underwent fMRI on both thighs before and immediately after 5 sets of 6 bilateral eccentric contractions of the 45° hip extension exercise or the Nordic exercise. Using fMRI, the transverse (T2) relaxation times were measured from pre-exercise and postexercise scans, and the percentage increase in T2 was used as an index of muscle activation. Results The fMRI revealed a significantly higher biceps femoris long head-to-semitendinosus ratio during the 45° hip extension exercise than in the Nordic exercise (P = .028). The T2 increase after the 45° hip extension exercise was greater for the biceps femoris long head (P<.001), semitendinosus, and semimembranosus (P≤.001) than that for the biceps femoris short head. During the Nordic exercise, the T2 increase of the semitendinosus was greater than that of the biceps femoris short head (P<.001) and biceps femoris long head (P = .001). Conclusion While both exercises involve high levels of semitendinosus activation in women, the Nordic exercise preferentially recruits that muscle, while the hip extension exercise more evenly activates all the biarticular hamstrings. J Orthop Sports Phys Ther 2018;48(8):607-612. Epub 23 Apr 2018. doi:10.2519/jospt.2018.7748.