Hamstring strain injuries (HSIs) represent the most common cause of lost playing time in rugby union. Eccentric knee flexor weakness and between-limb imbalance in eccentric knee flexor strength are associated with a heightened risk of HSIs in other sports; however, these variables have not been explored in rugby union.To determine if lower levels of eccentric knee flexor strength or greater between-limb imbalance in this parameter during the Nordic hamstring exercise are risk factors for HSIs in rugby union.Cohort study; Level of evidence, 2.This prospective study was conducted over the 2014 Super Rugby and Queensland Rugby Union seasons. In total, 178 rugby union players (mean age, 22.6 ± 3.8 years; mean height, 185.0 ± 6.8 cm; mean weight, 96.5 ± 13.1 kg) had their eccentric knee flexor strength assessed using a custom-made device during the preseason. Reports of previous hamstring, quadriceps, groin, calf, and anterior cruciate ligament injuries were also obtained. The main outcome measure was the prospective occurrence of HSIs.Twenty players suffered at least 1 HSI during the study period. Players with a history of HSIs had a 4.1-fold (95% CI, 1.9-8.9; P = .001) greater risk of subsequent HSIs than players without such a history. Between-limb imbalance in eccentric knee flexor strength of ≥15% and ≥20% increased the risk of HSIs by 2.4-fold (95% CI, 1.1-5.5; P = .033) and 3.4-fold (95% CI, 1.5-7.6; P = .003), respectively. Lower eccentric knee flexor strength and other prior injuries were not associated with an increased risk of future HSIs. Multivariate logistic regression revealed that the risk of reinjuries was augmented in players with strength imbalances.Previous HSIs and between-limb imbalance in eccentric knee flexor strength were associated with an increased risk of future HSIs in rugby union. These results support the rationale for reducing imbalance, particularly in players who have suffered a prior HSI, to mitigate the risk of future injuries.