Objective: This study aimed to identify the area with greatest effect using self-myofascial release technique (self-MFR) in the hamstring, suboccipital, and plantar regions. Design: Cross-sectional study. Methods: Twenty-two adult subjects were evaluated for flexibility and hamstring pain threshold after self-MFR. Based on the superficial back line, the self-MFR application areas were the suboccipital region, hamstring, and plantar regions. Self-MFR was applied to each area using a wooden pole for a total of 4 minutes. Self-MFR was applied for 3 days at the same time of day, which was randomly assigned for each subject. Treatment was applied to one area each day. The sit and reach test (SRT), active range of motion (AROM), and passive ROM (PROM) were used to determine changes in flexibility, and an algometer was used to determine pain threshold. Pre/post-self-MFR effectiveness was tested using a paired t-test. Repeated measurement was used to compare self-MFR effects in the suboccipital, hamstring, and plantar regions. Results: When the self-MFR technique was applied to the 3 areas, the SRT showed significant improvement over baseline (p<0.05). Bilateral AROM and PROM showed significant improvements (p<0.05). When the self-MFR technique was applied to the hamstring, the semimembranosus showed a significant change in pain threshold (p<0.05). Conclusions: Our findings suggest that indirect application based on the Anatomy Trains could be effective for those who need to improve muscle flexibility. Moreover, self-MFR easily alleviates myofascial pain while maintaining flexibility, and can be performed at any time and place.