Genetic ‘turnover’ — the extinction and replacement of biological diversity — represents a fascinating but poorly-understood issue in Southern Hemisphere prehistory. Here we present ancient DNA and radiocarbon analyses of archaeological remains to assess the chronologies and causes of prehistoric megafaunal extinction and replacement in southern New Zealand. The collated data include ancient DNA sequences from over 200 ancient sea-lion (Phocarctos) and penguin (Megadyptes) specimens, in addition to 150 modern genetic samples. Temporal genetic analyses show that sudden, synchronous megafaunal turnover events occurred in New Zealand at around 1500 AD, coinciding with the Little Ice Age onset and an associated drastic human demographic decline in the south of the country. We conclude that a combination of climatic and human demographic shifts facilitated northward expansion of subantarctic sea lion and penguin lineages, replacing extirpated mainland New Zealand megafauna. Broadly, the interaction between human pressure and late Holocene climatic change is implicated as a cause of multiple biological turnover events in the Southern Hemisphere.