It is believed that regional activation within a muscle may lead to greater site-specific muscular adaptations in the activated portion of the muscle. Because the hamstrings are a biarticular muscle, it can be theorized that single-joint exercises where movement originates at the hip vs. the knee will result in differential activation of the muscle complex. The purpose of the present study was to assess electromyographic activity in the proximal and distal aspects of the medial and lateral hamstrings during performance of the stiff-legged deadlift (SLDL), a hip-dominant exercise, and the lying leg curl (LLC), a knee-dominant exercise. Ten young, resistance-trained men were recruited from a university population to participate in the study. Employing a within-subject design, participants performed the SLDL and LLC to muscular failure using a load equating to their 8 repetition maximum for each exercise. The order of performance of exercises was counterbalanced between participants so that approximately half of the subjects performed SLDL first and the other half performed LLC first. Surface electromyography was used to record mean normalized muscle activity of the upper lateral hamstrings, lower lateral hamstrings, upper medial hamstrings, and lower medial hamstrings. Results showed that the LLC elicited significantly greater normalized mean activation of the lower lateral and lower medial hamstrings compared with the SLDL (p ≤ 0.05). These findings support the notion that the hamstrings can be regionally targeted through exercise selection. Further investigations are required to determine whether differences in activation lead to greater muscular adaptations in the muscle complex.