Botanical gardens have long contributed to plant science and have played a leading role in ex situ conservation, namely of threatened tree species. Focusing on the three botanical gardens of Lisbon (i.e., Botanical Garden of Ajuda—JBA, Lisbon Botanical Garden—JBL, and Tropical Botanical Garden—JBT), this study aims to reveal their natural heritage and to understand the historical motivations for their creation. Our results showed that these gardens contain a total of 2551 tree specimens, corresponding to 462 taxa, within 80 plant families. Of these, 85 taxa are found in the three gardens, and more than half of the taxa are hosted in JBL (334 taxa), whereas 230 and 201 taxa were recorded in JBT and JBA, respectively. The motivations for the creation of each garden are reflected in the different geographic origins of the trees they host in their living collections. The Palearctic species are dominant in JBA and JBL, and Tropical trees prevail in JBT. With more than 250 years of history, these gardens hold an invaluable natural and historical heritage, with their living collections providing valuable sources of information for the conservation of threatened plant species, at local and global scales.