Accumulating evidence demonstrates that pain induces adaptations in the corticomotor representations of affected muscles. However, previous work has primarily investigated the upper limb, with few studies examining corticomotor reorganization in response to lower limb pain. This is important to consider, given the significant functional, anatomical, and neurophysiological differences between upper and lower limb musculature. Previous work has also focused on unilateral corticomotor changes in response to muscle pain, despite an abundance of literature demonstrating that unilateral pain conditions are commonly associated with bilateral motor dysfunction. For the first time, this study investigated the effect of unilateral acute hamstring pain on bilateral corticomotor organization using transcranial magnetic stimulation (TMS) mapping. Corticomotor outcomes (TMS maps), pain, mechanical sensitivity (pressure pain thresholds), and function (maximal voluntary contractions) were recorded from 28 healthy participants at baseline. An injection of pain-inducing hypertonic (n = 14) or pain-free isotonic (n = 14) saline was then administered to the right hamstring muscle, and pain ratings were collected every 30 seconds until pain resolution. Follow-up measures were taken immediately following pain resolution and at 25, 50, and 75 minutes post-pain resolution. Unilateral acute hamstring pain induced bilateral symptom development and changes in corticomotor reorganization. Two patterns of reorganization were observed—corticomotor facilitation and corticomotor depression. Corticomotor facilitation was associated with increased mechanical sensitivity and decreased function bilaterally (all P < .05). These effects persisted for at least 75 minutes after pain resolution. PerspectiveThese findings suggest that individual patterns of corticomotor reorganization may contribute to ongoing functional deficits of either limb following acute unilateral lower limb pain. Further research is required to assess these adaptations and the possible long-term implications for rehabilitation and reinjury risk in cohorts with acute hamstring injury.