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In the Anthropocene, the world’s plant diversity is threatened with extinction and the erosion of the genetic diversity of natural populations. According to the State of the World’s Plants and Fungi 2020 of the Royal Botanic Gardens, Kew, two out of five of the ~350,000 known vascular plant species are at risk of extinction. Despite the considerable toolkit of biodiversity conservation practices, usually it is hard to choose the best option to stop biodiversity loss. Ex situ conservation has seen massive development due to radical losses of natural ecosystems, and its incrementing necessity has been underscored by Target 8 of the 2011-2020 Global Strategy for Plant Conservation. As we crossed the finish line of this strategy in 2020, a review of the accumulated knowledge on the ex situ living collections has become particularly important. Despite the increasing attention received by ex situ conservation, studies on the sustainability, quality, and usability of the plant material prior to establishing the garden collections are few, leaving major gaps unfilled in terms of best ex situ conservation practices. Here we present an overview of the results and experiences in ex situ conservation focusing on living plant collections, with the aim of guiding conservation practitioners towards the most efficient working methods. We evaluate the future needs and perspectives of this conservation technique, based on case studies on both woody and herb species. Possible conservation applications and priorities suggested for future works are summarized.