An alarming proportion of Australia's unique plant biodiversity is under siege from a variety of environmental threats. Options for in situ conservation are becoming increasingly compromised as encroaching land use, climate change and introduced diseases are highly likely to erode sanctuaries regardless of best intentions. Ex situ conservation is currently limited to botanic garden living collections and seed banking, with in vitro and cryopreservation technologies still being developed to address ex situ conservation of species not amenable to conventional storage. Cryopreservation (storage in liquid nitrogen) has been used successfully for long-term biosecure storage of shoot tips of several species of threatened Australian plants. We present a case for building on this research and fostering further development and utilisation of cryopreservation as the best means of capturing critical germplasm collections of Australian species with special storage requirements (e.g. recalcitrant-seeded taxa and species with short-lived seeds) that currently cannot be preserved effectively by other means. This review highlights the major issues in cryopreservation that can limit survival including ice crystal damage and desiccation, toxicity of cryoprotective agents, membrane damage, oxidative stress and mitochondrial function. Progress in understanding and mitigating these stresses is vital for advancing cryopreservation for conservation purposes.