Humans evolved in a world of giant creatures. Current evidence suggests that most ice age megafauna went extinct around the transition to our current Holocene epoch. The ecological reverberations associated with the loss of over 65% of Earth’s largest terrestrial animals transformed ecosystems and human lifeways forever thereafter. However, there is still substantial debate as to the cause of this mass extinction. Evidence variously supports climate change and anthropogenic factors as primary drivers in the restructuring of the terrestrial biosphere. Much of the ongoing debate is driven by the insufficient resolution accessible via macro-remains. To help fill in the gaps in our understandings of the Pleistocene-Holocene transition, I utilized the growing power of sedimentary ancient DNA (sedaDNA) to reconstruct shifting signals of plants and animals in central Yukon. To date, sedaDNA has typically been analyzed by amplifying small, taxonomically informative regions. However, this approach is not ideally suited to the degraded characteristics of sedaDNA and ignores most of the potential data. Means of isolating sedaDNA have also suffered from the use of overly aggressive purification techniques resulting in substantial loss. To address these limitations, I first experimentally developed a novel means of releasing and isolating sedaDNA. Secondly, I developed a novel environmental bait-set designed to simultaneously capture DNA informative of macro-scale ecosystems. When combined, we identify a substantial improvement in the quantity and breadth of biomolecules recovered. These optimizations facilitated the unexpected discovery of horse and mammoth surviving thousands of years after their supposed extirpation. I followed up these results by extracting DNA from multiple permafrost cores where we confirm the late survival signal and identify a far more complex and high-resolution dataset beyond those identifiable by complementary methods. I was also able to reconstruct mitochondrial genomes from multiple megafauna simultaneously solely from sediment, demonstrating the information potential of sedaDNA. / Dissertation / Doctor of Philosophy (PhD) / A new addition to the rapidly growing field of palaeogenetics is environmental DNA (eDNA) with its immense wealth of biomolecules preserved over millennia outside of biological tissues. Organisms are constantly shedding cells, and while most of this DNA is metabolized or otherwise degraded, some small fraction is preserved through sedimentary mineral-binding. I experimentally developed new ancient eDNA methods for recovery, isolation, and analysis to maximize our access to these biomolecules and demonstrate that this novel approach outperforms alternative protocols. Thereafter, I used these methods to extract DNA from ice age permafrost samples dating between 30,000–6,000 years before present. These data demonstrate the power of ancient eDNA for reconstructing ecosystem change through time, as well as identifying evidence for the Holocene survival of caballine horse and woolly mammoth in continental North America. This late persistence of Pleistocene fauna has implications for understanding the human ecological and climatological factors involved in the Late Pleistocene mass extinction event. This effort is paralleled with megafaunal mitogenomic assembly and phylogenetics solely from sediment. This thesis demonstrates that environmental DNA can significantly augment macro-scale buried records in palaeoecology.