A diverse assemblage of late Pleistocene marsupial trackways on a lake bed in south-western Victoria provides the first information relating to the gaits and morphology of several megafaunal species, and represents the most speciose and best preserved megafaunal footprint site in Australia. The 60-110 ka volcaniclastic lacustrine sedimentary rocks preserve trackways of the diprotodontid Diprotodon optatum, a macropodid (probably Protemnodon sp.) and a large vombatid (perhaps Ramsayia magna or 'Phascolomys' medius) and possible prints of the marsupial lion, Thylacoleo carnifex. The footprints were imprinted within a short time period, demonstrating the association of the taxa present, rather than the time-averaged accumulations usually observed in skeletal fossil deposits. Individual manus and pes prints are distinguishable in some trackways, and in many cases some digital pad morphology is also present. Several parameters traditionally used to differentiate ichnotaxa, including trackway gauge and the degree of print in-turning relative to the midline, are shown to be subject to significant intraspecific variation in marsupials. Sexual dimorphism in the trackway proportions of Diprotodon, and its potential for occurrence in all large bodied, quadrupedal marsupials, is identified here for the first time.