We used mtDNA and isozyme analysis of a freshwater fish, Galaxias divergens (Osmeriformes: Galaxiidae), to test a hypothesis of drainage evolution in South Island, New Zealand. Geological evidence indicates that the presently north-flowing Kaituna River branch of the Pelorus River system once flowed south into the Wairau River system. The subsequent flow-reversal is thought to have resulted from Pleistocene subsidence in central New Zealand. mtDNA sequence data corroborated this geological hypothesis: rivers draining into Pelorus Sound were found to retain a genetic lineage of G. divergens that is otherwise restricted to the Wairau River system and adjacent coastal drainages (based on current sampling). Other sampled drainages in northern South Island and southern North Island were found to house lineages that were highly divergent from the Wairau–Pelorus clade. Isozyme data yielded groupings based on fixed differences that were largely congruent with mtDNA clades. Standard molecular calibrations suggest that vicariant isolation of Pelorus and Wairau systems (drainage reversal) occurred in the mid-Pleistocene rather than the late Pleistocene as suggested by geology. Future multidisciplinary analyses will aim to improve our understanding of geological and molecular evolutionary rates. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88, 367–376.