Marginal populations are usually located at the edge of the species' range, constituting small isolates that grow in suboptimal environments. In widely distributed conifers, such as pines, marginal populations have often been considered recent or rare colonizing events without ecological relevance. The Canary Island pine provides an excellent case to study the significance of critically endangered marginal populations (from 20 to a few hundred trees) in an endemic conifer that is also extensively used in commercial plantations. Our chloroplast microsatellite (cpSSR) survey showed contrasting values of haplotypic genetic diversity in marginal populations of Canary island pine, from very low at Garabato (northern La Gomera) and Anaga (Tenerife), located within broad-leaved tree formations, to very high in Gran Canaria marginal populations (Arguineguin and Galdar). Competition with broad-leaved vegetation, after rare long-distance colonization events, might explain present-day low levels of variation in Garabato and Anaga. Gran Canaria and southern La Gomera relict populations, which contain high haplotypic diversity, are probably remnants of larger populations severely reduced by adverse climatic conditions and human activities, the latter starting shortly after the European colonization of the islands in the XV century. Given the extremely low population numbers and a general lack of natural regeneration, the risk of extinction in the near future is high in marginal populations of Canary Island pine. Conservation activities are urgently required and should focus on the highly diverse Gran Canaria and southern La Gomera pine marginal populations. Here, the establishment of in situ genetic reserves (Arguineguin and Imada), and multifunctional ex situ collections (Arguineguin and Galdar), such as seed orchards, is highly recommended. In addition, common garden experiments to test the performance of potentially drought-tolerant genotypes from Arguineguin (southernmost Gran Canaria's relict) and potential risks due to inbreeding and/or outbreeding depression should be established.