Australian football requires frequent intermittent sprinting close to peak running speed. However, tests assessing the capability to maintain intermittent peak running speed are not reported in scientific literature. Therefore, our objective is to report the reliability and validity of a novel intermittent peak running speed test. The intermittent peak running speed test required footballers to perform 10 repetitions on 25-second intervals. Each repetition required 15-m jogging, 20-m acceleration to peak speed, 10 m to sustain peak speed, 20-m deceleration, and finally a 15-m jog. Intermittent peak running speed was determined by portable global positioning system. To assess reliability, 26 footballers performed the intermittent peak running speed test on 2 occasions 3-5 days apart. Our results revealed that average peak speed had a coefficient of variation of 2.2% and an intraclass correlation of 0.91. To assess construct validity, average peak speed was compared between elite, sub-elite, and regional footballers. The average peak speed of the elite footballers (28.6 ± 1.7 km · h(-1)) was higher than that of the sub-elite (27.4 ± 1.7 km · h(-1)) and regional (27 ± 1.9 km · h(-1)) competitors (p < 0.05). Our study revealed that the intermittent peak running speed test possesses acceptable reliability and distinguishes between elite and sub-elite footballers.