A purpose of this study was to determine if pre-season anthropometric and physiological measures were significantly different for the players from one Australian Football League (AFL) club selected to play in the first game of the season compared to the players not selected. Another purpose was to compare fitness test results for defenders, forwards and mid-fielders in the same AFL club. Thirty-four players were tested for isolated quadriceps and hamstrings strength, leg extensor muscle strength and power, upper body strength, sprinting speed, vertical jump (VJ), endurance, skinfolds and hamstring flexibility. The starters who were selected to play the first game were a significantly older and more experienced playing group, and were significantly better (p < 0.05) in measures of leg power, sprinting speed and the distance covered in the Yo Yo intermittent recovery test compared to the non-starters. Although there were trends for the superiority of the starters, the differences in lower and upper body strength, VJ and predicted VO(2)max were nonsignificant. The forwards generally produced the worst fitness scores of the playing positions with the midfielders having significantly lower skinfolds and the defenders possessing better hamstring strength and VJ compared to the forwards. It was concluded that some fitness qualities can differentiate between starters and non-starters, at least in one AFL club. Comparisons of playing positions and the development of fitness norms for AFL players require further research.