Understanding factors driving diversity across biodiversity hotspots is critical for formulating conservation priorities in the face of ongoing and escalating environmental deterioration. While biodiversity hotspots encompass a small fraction of Earth’s land surface, more than half the world’s plants and two-thirds of terrestrial vertebrate species are endemic to these hotspots. Tropical Southeast Asia displays extraordinary species richness, encompassing four biodiversity hotspots, though disentangling multiple potential drivers of species richness is confounded by the region’s dynamic geological and climatic history. Here, we use multi-locus molecular genetic data from dense multi-species sampling of freshwater fishes across three biodiversity hotspots, to test the effect of Quaternary climate change and resulting drainage rearrangements on aquatic faunal diversification. While Cenozoic geological processes have clearly shaped evolutionary history in Southeast Asian halfbeak fishes, we show that paleo-drainage re-arrangements resulting from Quaternary climate change played a significant role in the spatiotemporal evolution of lowland aquatic taxa, and provides priorities for conservation efforts.