Background: The hamstring muscles are described as forming part of myofascial chains or meridians, and the superficial back line (SBL) is one such chain. Good hamstring flexibility is fundamental to sporting performance and is associated with prevention of injuries of these muscles. The aim of this study was to measure the effect of self-myofascial release (SMR) on hamstring flexibility and determine which segment of the SBL resulted in the greatest increase in flexibility. Methods: 94 volunteers were randomly assigned to a control group or to one of the five intervention groups. In the intervention groups, SMR was applied to one of the five segments of the SBL (plantar fascia, posterior part of the sural fascia, posterior part of the crural fascia, lumbar fascia or epicranial aponeurosis) for 10 min. The analyzed variables were hamstring flexibility at 30 s, 2, 5, and 10 min, and dorsiflexion range of motion before and after the intervention. Results: Hamstring flexibility and ankle dorsiflexion improved when SMR was performed on any of the SBL segments. The segments with the greatest effect were the posterior part of the sural fascia when the intervention was brief (30 s to 2 min) or the posterior part of the crural fascia when the intervention was longer (5 or 10 min). In general, 50% of the flexibility gain was obtained during the first 2 min of SMR. Conclusions: The SBL may be considered a functional structure, and SMR to any of the segments can improve hamstring flexibility and ankle dorsiflexion.