Strategies directed at the prevention of disabling pain have been suggested as a public health priority, making early identification of youth at risk for poor outcomes critical. At present, limited information is available to predict which youth presenting with acute pain are at risk for persistence. The aims of this prospective longitudinal study were to identify biopsychosocial factors in the acute period that predict the transition to persistent pain in youth with new-onset musculoskeletal (MSK) pain complaints. Participants were 88 children and adolescents (age 10-17 years) presenting to the emergency department (n = 47) or orthopedic clinic (n = 41) for evaluation of a new MSK pain complaint (<1 month duration). Youth presented for 2 study visits (T1 ≤1 month post pain onset; T2 = 4-month follow-up) during which they completed questionnaires (assessing pain characteristics, psychological factors, sleep quality) and participated in a laboratory task assessing conditioned pain modulation. Regression analyses tested T1 predictors of longitudinal pain outcomes (pain persistence, pain-related disability, quality of life QOL). Results revealed approximately 35% of youth had persistent pain at 4-month follow-up, with persistent pain predicted by poorer conditioned pain modulation and female sex. Higher depressive symptoms at T1 were associated with higher pain-related disability and poorer QOL at T2. Findings highlight the roles of depressive symptoms and pain modulation in longitudinally predicting pain persistence in treatment-seeking youth with acute MSK pain and suggest potential mechanisms in the transition from acute to chronic MSK pain in children and adolescents.