Context Distribution and abundance of the three northern Australian tree-rat species, Conilurus penicillatus, Mesembriomys macrurus and M. gouldii, have contracted significantly since European settlement. All three species were recorded from the Mitchell Plateau, Western Australia, in the 1980s, enabling their niche separation to be assessed. Despite this study being conducted in 1987, the results remain relevant for conservation strategies for these species. Aims To determine the ecological separation among three tropical tree-rat species by documenting habitat preferences, habitat use and diet. Methods Live-trapping, radiotracking and spotlighting were used to assess relative abundance, habitat preference and movements of each species at nine sites on the Mitchell Plateau. Diet was determined using scat analysis; vegetation communities were described and characterised by pattern analysis. Key results C. penicillatus (number of individuals trapped (n) = 24) was most abundant, followed by M. macrurus (n = 8) and M. gouldii (n = 2). Mature open forest with a well-developed understorey was preferred by the predominantly arboreal M. gouldii (1 site), M. macrurus occupied more varied habitats (four sites), especially ecotones between vine thicket and open eucalypt forest and the boulder edges of the plateau, and C. penicillatus occupied the greatest variety of habitats (four sites) in open eucalypt woodland with a diverse shrub layer and ground cover almost absent to very dense, tall grass. Diet analysis identified M. gouldii as a frugivore (fruits, some flowers), M. macrurus as an omnivore (seeds, fruits, flowers, termites, other invertebrates), and C. penicillatus as a generalist (monocot and dicot leaves, seeds, flowers, fruits, some arthropods). Conclusions The largest of the species, M. gouldii, appeared to have the narrowest ecological niche, preferring more stable, structurally diverse, mature open forest that is infrequently burnt. The mid-sized M. macrurus occupies a broader range of habitats, particularly ecotones between vine thickets and eucalypt forest/woodland and woodland. C. penicillatus preferred open eucalypt woodland with a variable understorey. Implications With a changing climate, increased fire frequency, habitat degradation by introduced herbivores and predation by cats across the tropical savanna distribution of these threatened tree-rats, this study provides data to underpin actions that may ensure their survival.