Recent studies suggest region-specific metabolic activity in hamstring muscles during injury prevention exercises, but the neural representation of this phenomenon is unknown. The aim of this study was to examine whether regional differences are evident in the activity of biceps femoris long head (BFlh) and semitendinosus (ST) muscles during two common injury prevention exercises. Twelve male participants without a history of hamstring injury performed the Nordic hamstring exercise (NHE) and stiff-leg deadlift (SDL) while BFlh and ST activities were recorded with high-density electromyography (HD-EMG). Normalized activity was calculated from the distal, middle, and proximal regions in the eccentric phase of each exercise. In NHE, ST overall activity was substantially higher than in BFlh (d = 1.06 ± 0.45), compared to trivial differences between muscles in SDL (d = 0.19 ± 0.34). Regional differences were found in NHE for both muscles, with different proximal-distal patterns: The distal region showed the lowest activity level in ST (regional differences, d range = 0.55-1.41) but the highest activity level in BFlh (regional differences, d range = 0.38-1.25). In SDL, regional differences were smaller in both muscles (d range = 0.29-0.67 and 0.16-0.63 in ST and BFlh, respectively) than in NHE. The use of HD-EMG in hamstrings revealed heterogeneous hamstrings activity during typical injury prevention exercises. High-density EMG might be useful in future studies to provide a comprehensive overview of hamstring muscle activity in other exercises and high-injury risk tasks.