Leg stiffness is a modifiable mechanical property that may be related to soft tissue injury risk. The purpose of this study was to examine mean leg stiffness and bilateral differences in leg stiffness across an entire professional Australian Football League (AFL) season, and determine whether this parameter was related to the incidence of lower body soft tissue injury. The stiffness of the left and right legs of 39 professional AFL players (age 24.4 ± 4.4 years, body mass 87.4 ± 8.1 kg, stature 1.87 ± 0.07 m) was measured using a unilateral hopping test at least once per month throughout the season. Injury data were obtained directly from the head medical officer at the football club. Mean leg stiffness and bilateral differences in leg stiffness were compared between the injured and non-injured players. There was no difference between the season mean leg stiffness values for the injured (219.3 ± 16.1 N x m(-1) x kg(-1)) and non-injured (217.4 ± 14.9 N x m(-1) x kg(-1); P = 0.721) groups. The injured group (7.5 ± 3.0%) recorded a significantly higher season mean bilateral difference in leg stiffness than the non-injured group (5.5 ± 1.3%; P = 0.05). A relatively high bilateral difference in leg stiffness appears to be related to the incidence of soft tissue injury in Australian football players. This information is of particular importance to medical and conditioning staff across a variety of sports.