The relict character of the Scots pine populations from the Tatra Mts. (Western Carpathians) was formed on scarcely accessible, ecologically extreme habitats, in areas which were not under human activity. An exhaustive genetic analysis of the Scots pine populations from the refugial locations in the Tatra Mts. (Poland) had not yet been. In this study, we characterize the genetic variation and differentiation of the relict Scots pine populations from the Tatra Mts., to provide information on their genetic resources and the conservation implications. Eight paternally inherited chloroplast microsatellite loci were used to investigate the genetic structure of 4 relict populations, which were compared with the natural populations from the Baltic region (5 populations from Lithuania, Latvia and Estonia) and to a northern range from Russia (5 populations from the Kola Peninsula and Arkhangelsk Oblast). The four relict pine populations were characterized by a lower level of genetic variation when compared to the Baltic and northern populations, which was expressed in lower values of particular genetic parameters: numbers of alleles and haplotypes, haplotype genetic diversity, haplotype differentiation, and mean genetic distance between individuals in the population. Our results revealed a very high and significant genetic differentiation between all the analyzed populations, as well as between the three analyzed regions (PhiPT = 8%). Furthermore, the relict populations within a close geographic location showed higher differentiation (PhiPT = 5%) compared to the northern Russian and Baltic populations that were separated by tens and hundreds of kilometers. The relict populations of P. sylvestris from the Tatra Mts. deserve conservation efforts due to the fact that they are an important component if the species-poor Tatra forests.