a b s t r a c t Fossils and sediments preserved in caves are an excellent source of information for investigating impacts of past environmental changes on biodiversity. Until recently studies have relied on morphology-based palaeontological approaches, but recent advances in molecular analytical methods offer excellent po-tential for extracting a greater array of biological information from these sites. This study presents a thorough assessment of DNA preservation from late PleistoceneeHolocene vertebrate fossils and sedi-ments from Kelly Hill Cave Kangaroo Island, South Australia. Using a combination of extraction tech-niques and sequencing technologies, ancient DNA was characterised from over 70 bones and 20 sediment samples from 15 stratigraphic layers ranging in age from >20 ka to w6.8 ka. A combination of primers targeting marsupial and placental mammals, reptiles and two universal plant primers were used to reveal genetic biodiversity for comparison with the mainland and with the morphological fossil record for Kelly Hill Cave. We demonstrate that Kelly Hill Cave has excellent long-term DNA preservation, back to at least 20 ka. This contrasts with the majority of Australian cave sites thus far explored for ancient DNA preservation, and highlights the great promise Kangaroo Island caves hold for yielding the hitherto-elusive DNA of extinct Australian Pleistocene species.