BACKGROUND: Hamstring strains remain one of the most prevalent injuries in Australian Rules football. The authors prospectively examined the relationship between musculotendinous stiffness of the hamstring and leg stiffness with hamstring injury in professional Australian Rules footballers during the 2006 season. HYPOTHESIS: Higher hamstring stiffness and leg stiffness are related to noncontact, soft tissue hamstring injury risk in professional Australian Rules footballers. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: Unilateral hamstring stiffness and leg stiffness were assessed in 136 professional footballers in the month before the commencement of the competitive season. This information was then investigated relative to players who suffered noncontact, soft tissue hamstring injuries during either matches or training throughout the season to identify whether preseason stiffness was related to injury occurrence. RESULTS: Fourteen tested players recorded acute, noncontact hamstring injuries, resulting in 3.3 ± 2.8 weeks of missed match play per injury. At preseason testing, the players who ended up sustaining a hamstring injury during the season recorded significantly higher mean hamstring stiffness (11%, P = .04) and leg stiffness (5%, P = .03). When considering the injured players, the leg stiffness of the involved limb was significantly higher than the noninjured players (P = .02), whereas hamstring stiffness was significantly higher on the noninvolved limb (P = .01). Further, those players who suffered a hamstring injury were significantly older than the noninjured players (P = .01). CONCLUSION: It appears that a high bilateral hamstring stiffness and leg stiffness may be a determinant in the risk of sustaining a hamstring injury. Further, relatively lower hamstring stiffness in the involved limb of injured players appears to be associated with increased injury and may be related to a lack of strength. The information from stiffness assessment may allow medical staff to determine the hamstring risk status for individual players in team sports.