Before it became extinct in 1936, the Tasmanian tiger or thylacine (Thylacinus cynocephalus) was the world’s largest carnivorous marsupial. Its extinction from mainland Australia approximately 3000 years ago, and survival on the island of Tasmania, is a mystery that has attracted much debate and research. The timing of the thylacines’ mainland extinction implicates the introduction of the dingo, human intensification and climate change as possible causes. However, extinction time alone is insufficient to resolve the relative importance of these extinction drivers, and determining past population dynamics is essential for this debate to move forward. For example, recent genetic research on Tasmanian and mainland devils (Sarcophilus harrisii, which also became extinct on the mainland 3000 years ago), suggests that their mainland extinction was synchronous with a severe decline in the Tasmanian population, implicating a common driver. Here we present the first ancient DNA sequences from mainland thylacines and compare these to the recently extinct Tasmanian population. We used hybridization capture and next generation sequencing to generate mitochondrial genomes from ancient mainland (>3290 years before present), ancient Tasmanian (>500 years before present), and historic Tasmanian (1800-1936) thylacines. We use these data to examine the demographic history of thylacines on Tasmania and the mainland and gain insight into what led to the mainland extinction and Tasmanian survival.