Twenty-one populations of Scots pine sampled over the entire Italian range of the species were analysed for genetic variation scored at nine nuclear SSR markers. The main aim of the work was to find genetic features seful for conservation management, namely allelic composition, gene diversity and differentiation. High levels of intra-population variability were scored. The only population sampled in the Apennines gave the lowest values, confirming the genetic erosion undergone in the Scots pine remnants in this area. A low level of genetic variability was also scored for populations from the Po valley and hills of Piedmont. Most genetic diversity was found within populations, while only a small amount occurred among them (FST = 0.058). Both Bayesian clustering and sPCA analysis showed a East–West subdivision, notwithstanding the unclear position of populations from the Po valley. The population from the Apennines was always clearly separated from the others. The results are discussed in terms of post-glacial recolonisation, as well as for defining genetically homogeneous regions for Scots pine in Italy. The management of genetic resources could benefit from the identification of such ‘gene zones’, thereby avoiding the use of non-local reproductive material for plantations, which can represent one of the most important reasons for failure of reforestation. In addition, the assessment of the biogeographic genetic structure by neutral markers is a prerequisite for disentangling the influence of selectively neutral and non-neutral processes on the distribution of adaptive genetic variability.