The origin of high diversity in tropical rainforests is attributed to divergence amongst relatively mesic, late Pleistocene refuges. To test this hypothesis we analysed sequence variation within the mitochondrial DNA of populations of six rainforest-dwelling vertebrate species (one lizard and five birds) in the Wet Tropical rainforests of northeastern Australia. Vicariance among historical refuges was indicated by geographically congruent variation across a historical climatic barrier previously predicted by modelling. Sequence divergence across the barrier varied widely, being highest in species with lowest vagility and greatest restriction to rainforest. A high altitude, rainforest-restricted species was exceptional in lacking mtDNA variation. These data support the suggested role of vicariance in promoting evolutionary diversity in rainforests but also indicate variation in the timing of vicariance events, with most speciation or divergence events occurring well before the late Pleistocene. The relation between the amount of molecular divergence and current ecology suggests a sequence of isolation events, perhaps of increasing severity, occurring between the late Tertiary and the late Pleistocene.