Objectives: Australian Football (AF) is a collision sport containing high injury rates in junior competition. Successful performance at the elite junior level not only requires superior specific football knowledge and skills, but also well developed fitness qualities. However, no studies have examined the link between physical fitness qualities and injury in AF. Design: Prospective cohort. Methods: Injury data were collected through the use of a Player Movement Record (PMR) and a standardized Injury Report Form (IRF). Fitness test data was collected during the pre-season of the 2010 and 2011 seasons. Results: 382 players consented to participate in the study. The cohort experienced an injury incidence rate of 24.29/standardized club (40 players/club). A faster 5-m sprint was associated with 'injury status' (p = 0.016) and a 'knee' region (p ≤ 0.001) injury. A faster planned agility score was associated with an increased risk of a 'hip/groin/thigh' region (p = 0.010) injury, and specifically a 'quadriceps strain' (p = 0.005). A lower 20-m shuttle run was associated with an increased risk of a 'shin/ankle/foot' (p = 0.045) injury. Increased injury severity was associated with a higher left foot running vertical jump (VJ) (p = 0.040), and faster 5-m sprint (p = 0.043). Conclusions: Lower aerobic endurance, faster 5-m acceleration and greater planned agility were associated with an increased risk of various injury types in elite junior AF players. Furthermore, a higher left foot running VJ and faster 5-m acceleration were associated with injury severity. These results may largely relate to a greater work capacity placing a higher load upon the musculoskeletal system in contact and non-contact situations.