BACKGROUND: Hamstring injuries are the most common injury sustained by elite Australian football players and result in substantial costs because of missed training time, unavailability for matches and lost player payments. Evidence to support proposed risk factors for hamstring injury is generally lacking, limiting the development of appropriate prevention strategies. AIM: To identify intrinsic risk factors for hamstring injury at the elite level of Australian football. METHODS: A prospective cohort of 222 players underwent baseline measurement in the form of a self-report questionnaire and a musculo-skeletal screen during the pre-season period of the 2002 Australian football season. Injury surveillance and exposure data were collected for the full season. Logistic regression analyses were used to identify independent predictors of hamstring injury in this group of players. RESULTS: Thirty-one players sustained a hamstring injury. A past history (previous 12 months) of hamstring injury and increasing age were found to be independent predictors of hamstring injury. CONCLUSIONS: Older players and those with a previous history of hamstring injury are target groups for further research and implementation of injury prevention strategies. Restricted ankle dorsiflexion range of movement warrants consideration in the development of prevention programs for hamstring injury.