This study was designed to determine the regional differences of magnetic resonance (MR) measurements in the hamstrings [biceps femoris muscle long head (BFl) and short head (BFs), semimembranosus muscle (SM), and semitendinosus muscle (ST)] following eccentric knee-flexion exercise. Twelve male volunteers performed eccentric knee-flexion exercise. Maximum isometric torque, plasma creatine kinase (CK) activity, muscle soreness, and MR images of the hamstrings were measured before and immediately following exercise, and on the first, second, third and seventh days following the exercise. Cross-sectional areas (CSAs) and transverse relaxation times (T2s) of the hamstrings were measured from the T2-weightened MR imaging sequences of 30% (proximal), 50% (middle), and 70% (distal) areas of the thigh length. The CSA of the ST at proximal and middle regions had significantly increased on the third day, but no significant changes were found for the BFl or SM. Immediately following exercise, T2 values had increased significantly in the BFs, BFl, and ST. On the third day, T2 values of only ST increased significantly at proximal, middle and distal regions. Significant differences of T2 values between proximal and distal regions in the ST were found for the second, third and seventh days following the exercise. These results suggest that because of the anatomical characteristics of the muscles, the degrees of response following the exercise differed among the muscles and the regions of ST muscle.