Objectives To investigate the relationship between physical qualities and match activity profiles of recreational Australian football players. Design Prospective cohort study. Methods Forty players from three recreational Australian football teams (Division One, Two and Three) underwent a battery of fitness tests (vertical jump, 10 and 40m sprint, 6m×30m repeated sprint test, Yo-Yo intermittent recovery level Two and 2-km time trial). The activity profiles of competitive match-play were quantified using 10-Hz Global Positioning System units. Results Division One players possessed greater maximum velocity, Yo-Yo level Two and 2-km time trial performances than Division Two and Three players. In addition, Division One players covered greater relative distance, and relative distances at moderate- and high-intensities during match-play than Division Two and Three players. Division Two players had better 2-km time trial performances than Division Three players. Positive associations (P <0.05) were found between 10m acceleration, maximum velocity, Yo-Yo level Two and 2-km time trial performances and relative distance, and relative distances covered at moderate- and high-intensities during match-play. Moderate relationships were found between vertical jump and relative distance and high-intensity running. Conclusions Sub-elite Australian football players competing at a higher level exhibit greater physical qualities and match-play activity profiles than lesser-skilled recreational players. Acceleration and maximum velocity, 2-km time trial and Yo-Yo level Two performances discriminate between players of different playing levels, and are related to physical match performance in recreational Australian football. The development of these qualities is likely to contribute to improved match performance in recreational Australian football players.