Background: Hamstring muscle tightness contributes to disability in people with chronic low back pain (CLBP). HM stretching improves flexibility in healthy individuals, but the immediate effect of stretching is unknown in people with CLBP. Moreover, the stretching effect could be influenced by psychosocial factors.Objectives: To evaluate the immediate effect of passive HM stretching on flexibility in people with CLBP and the relationships between psychosocial factors and change in hamstring flexibility. Design: Non-randomized, pilot trial.Method: One minute of passive stretching was performed in 90 people with CLBP. Change in Active Knee Extension and Straight Leg Raise angles (digital inclinometer), and Fingertips-to Floor distance (measuring tape) were measured before and immediately after stretching. Correlations between change in flexibility and baseline Fear-Avoidance Beliefs Questionnaire (FABQ) and Hospital Anxiety and Depression Scale (HADS) scores were analyzed.Results: Hamstring flexibility improved significantly after stretching; Active Knee Extension mean difference was 4 (95% CI, 2.4 to 5.1; p < 0.001, right ES = 0.24, left ES = 0.23); Straight Leg Raise mean difference was 7 (95% CI, 5.5 to 8.6, p < 0.001, right ES = 0.44, left ES = 0.42), Fingertips-to-Floor mean difference was 2 cm (95% CI, 1.7 to 3.0, p < 0.001, ES = 0.20). No correlation was found between improvement in any of the hamstring flexibility measurements and FABQ or HADS scores (p > 0.05).Conclusions: Passive hamstring stretching induced an immediate, statistically significantly improvement in hamstring flexibility, but only the change in Straight Leg Raise amplitude was clinically important. Psychosocial factors were not related to improvements in flexibility after hamstring stretching.