The Yo-Yo Intermittent Recovery (IR) Test is currently used to assess endurance performance in team sport athletes. However, to date, no data has been presented on its application to an elite junior Australian football (AF) playing group. Therefore, the aim of this study was to evaluate the Yo-Yo Intermittent Recovery Test Level 1 (IR1) ability to discriminate between junior AF players at two different playing standards and a group of non-athletic healthy males. Sixty age matched participants (16.6+/-0.5 years) spread over three groups (20 per group): elite junior footballers; sub-elite junior footballers; and non-athletic healthy males participated in this study. Participants undertook a single Yo-Yo test performance on an indoor basketball court for each group. A one-way ANOVA with Scheffe's post hoc analysis revealed the elite junior footballers covered a significantly greater total distance (p<0.001) and completed a significantly greater number of high-intensity efforts (p<0.001) in comparison to their sub-elite counterparts, whilst both AF groups performed significantly better (p<0.001) than the non-athletic healthy males. This study demonstrates the ability of the Yo-Yo IR1 to discriminate endurance performance between elite and sub-elite AF players, whilst further distinguishing AF players from a non-athletic healthy control group.