All eukaryote populations accumulate mutations in their mitochondrial DNA (mtDNA) over time, so reproductively isolated populations become characterized by distinct mtDNA lineages. In addition, the degree of genetic differentiation among distinct populations can be used to estimate time elapsed since their isolation. We have identified an informative system for calibrating the mtDNA “clock” by genetically comparing freshwater galaxiid fish populations isolated in different river drainages. Calibration using a range of Quaternary geological events in southern New Zealand shows that the mtDNA divergence rate in galaxiid fishes is between 1% and 2%/100 k.y. up to 250 k.y., with the rate decreasing with increasing age. The estimated divergence rate slows to around 4%/m.y. for the middle Quaternary, although calibration is poor. A calibration curve has been fitted to all data: divergence (%) = −2.2e−9t + 2.5t + 2.2, where t is isolation age (in m.y.). This molecular clock has potential as a dating tool for glacially related and active tectonic events that have caused river drainage changes in the late Quaternary in the Southern Hemisphere, where galaxiids are widespread. An application of this dating tool to an example in northern South Island uses three different species of freshwater-limited fish, and all three data sets imply formation of a drainage divide at 320 ± 110 ka, at about the time of a major glacial advance though the divide (oxygen isotope stage 8).