Background and Objective: The Hamstring muscles are found to be the most prevalent for the tightness in the body. Tightness of these muscles produces decrease range of motion and reduced flexibility of the pelvis, hip and knee joints. Muscle stiffness of the lower extremities and the consequential decrease in joint flexibility are considered to be major aetiological factors in musculoskeletal injuries. The purpose of this study is to compare the immediate effect of Muscle energy technique and Eccentric training on hamstring tightness of healthy female volunteers. Methods: Sixty healthy female volunteers in the age group of 18 to 22 years with bilateral hamstring tightness without any previous musculo skeletal injuries were randomly divided in to Muscle energy technique group and Eccentric training group with 30 each. A total of 120 hamstrings were treated as pre the intervention plan in both groups. The outcome measure was active knee extension range of motion measured in supine lying with hip flexed to 90 degrees by using universal goniometer. Results: The mean __ampersandsignplusmn; SD values in muscle energy technique group before intervention is 128.13__ampersandsignplusmn;5.33 and after intervention is 138.07__ampersandsignplusmn;5.51 with p__ampersandsignlt;0.001 and in eccentric training group before intervention is 132.27__ampersandsignplusmn;6.73 and after intervention is 137.33__ampersandsignplusmn;6.63 with p__ampersandsignlt;0.001 shows significant improvement in both groups. Intergroup comparison shows no significant difference between groups with p__ampersandsigngt;0.05. Conclusion: There is no difference between the immediate effect of Muscle energy technique and Eccentric training in lengthening the tight hamstring muscles of healthy females.