Genetic Diversity and Population Structure of Black Pine (Pinus nigra Arn.) in Mt. Athos, Northern Greece

European black pine (Pinus nigra Arn. subsp. nigra) persists in scattered montane stands across Greece, where isolated populations harbour genetic variation shaped by local environments and demographic history. In this study, we assessed the genetic diversity and population structure of P. nigra using nuclear microsatellite markers (nSSRs) across four populations: Mt. Athos, Sithonia, Thassos, and Perama. A total of 67 individuals were genotyped, and seven high-quality polymorphic loci were retained after rigorous filtering. The Mt. Athos population exhibited the highest allelic richness and heterozygosity, with all loci being polymorphic and a low inbreeding coefficient after null allele correction. In contrast, the Perama population displayed reduced diversity, fewer polymorphic loci, and persistent heterozygote deficits. Principal Component Analysis (PCA) and Discriminant Analysis of Principal Components (DAPC) revealed weak overall population structure, with Perama genetically distinct from the other sites. Spatial Principal Component Analysis (sPCA) further uncovered an east–west cline within Athos and localized structure potentially shaped by both natural isolation and human influence. These findings highlight regional variation in genetic diversity within P. nigra and identify Athos as a genetically rich population of particular interest. The results provide a foundation for long-term monitoring and support informed strategies for the management and conservation of P. nigra in Greece.