BACKGROUND: Hamstring muscle strain injuries (HMSI) are the greatest injury problem in kicking sports such as Australian Rules Football. Reduced hamstring muscle strength is commonly perceived to be a risk factor for hamstring injury; however, evidence is inconclusive. Testing hamstring strength with the hip and knee at functional angles and assessing endurance parameters may be more relevant for examining the risk of hamstring injury. OBJECTIVE: The primary aim of this prospective study was to examine if reduced hamstring muscle strength assessed with the single leg hamstring bridge (SLHB) was a risk factor for hamstring injury. METHODS: Hamstring muscle strength of 482 amateur and semielite players from 16 football clubs, mean age 20.7 (range 16-34 years), was tested during the 2011 preseason. Players were then monitored throughout the 2011 playing season for HMSI. RESULTS: A total of 28 hamstring injuries, 16 right and 12 left, were recorded. Players who sustained a right HMSI during the season had a significantly lower mean right SLHB score (p=0.029), were older (p=0.002) and were more likely to have sustained a past right hamstring injury (p=0.02) or right knee injury (p=0.035). For left-sided hamstring injury, the injured group was more likely to be left leg dominant (p=0.001), older athletes (p=0.002) and there was a trend towards a history of left hamstring injury (p=0.07). CONCLUSIONS: This study demonstrated a significant deficit in preseason SLHB scores on the right leg of players that subsequently sustained a right-sided hamstring injury. Age, previous knee injury and a history of hamstring injury were other risk factors supported in this study. Low hamstring strength appears to be a risk factor for hamstring injury; however, due to the confounding variables and low injury rate in this study, further studies are required.