Genetic diversity is essential to evolution and a recognized target of conservation. When threats are high and populations are small, in-situ gene conservation needs to be reinforced with an ex-situ approach, where a genetically representative sample of the target taxon is safeguarded in a favorable environment. The fragmented habitat of Pinus nigra subsp. salzmannii (Dunal) Franco 1943 (Salzmann’s pine) is threatened by wildfires. In France, gene flow from massive plantations of other subspecies of black pines threatens its genetic diversity. Here, using individual tree genotypic data at thirteen microsatellite loci, we identified differentiated lineages for in-situ gene conservation. Discriminating between autochthonous and hybrid trees, we proposed a method for the creation of an ex-situ core collection. We confirmed that Salzmann’s pine is an original genetic lineage within the western European and Mediterranean black pine subspecies. We identified five genetic groups in France that can serve as the basis for in-situ gene conservation. Maximizing overall genetic diversity while maintaining among population diversity, we identified 80 native and non-hybridized trees that can form the basis of a representative ex-situ core collection. Our cost-effective methods combining in-situ and ex-situ conservation can be easily applied to many forest tree species.