Aim The Tasmanian tiger, or thylacine, is an infamous example of a recent human-mediated extinction. Confined to the island of Tasmania in historical times, thylacines were hunted to extinction <150 years after European arrival. Thylacines were also once widespread across the Australian mainland, but became extinct there c. 3,200 years before present (bp). Very little is known about thylacine biology and population history; the cause of the thylacines extirpation from the mainland is still debated and the reasons for its survival in Tasmania into the 20th century are unclear. In this study, we investigate the thylacine's phylogeography and demographic history leading up to their extinction on both the mainland and Tasmania to gain insight into this enigmatic species. Location Southern Australia. Methods We generated 51 new thylacine mitochondrial DNA (mtDNA) genome sequences from sub-fossil remains and historical museum specimens, and analysed them to reconstruct the species’ phylogeography and demographic history. Results We found evidence that thylacines had contracted into separate eastern and western populations prior to the Last Glacial Maximum (c. 25,000 yr bp), and that the ancient western population was larger and more genetically diverse than the historical Tasmanian population. At the time of European arrival in c. 1800 CE, Tasmanian thylacines had limited mtDNA diversity, possibly resulting from a bottleneck event broadly coincident with an El Niño-Southern Oscillation (ENSO) associated climate event, although we find some indication that the population was expanding during the late Holocene. Main Conclusions The timing of this putative expansion, in concert with a climate event, suggests that climate change had an influence on thylacine population dynamics. Given that ENSO effects are known to have been more severe on mainland Australia, we suggest that climate change, in synergy with other drivers, is likely to have contributed to the thylacine mainland extinction.