The present state of knowledge regarding the genetic diversity of forest tree species has been greatly improved with the development of the powerful research tool that the microsatellite markers represent. These noncoding sequences are considered to be neutral, highly polymorphic, and species specific. The usefulness of the microsatellite markers was recently proven by the determination of differentiation at inter-and intrapopulation level, gene flow in natural forest-tree populations, heritability processes, and sustainable management of forest genetic resources in many natural forest stands. In this chapter, I aim to describe the practical approach of microsatellite markers, used in determination of genetic structure of 14 Scots pine populations from North-eastern Poland. Investigated pine populations exhibited high genetic parameter variation, for example, mean PIC = 79.3, Shannon Index I = 2.488, observed (H O = 0.778) and expected (H E = 0.849) heterozygosity. Low level of F st = 0.031 demonstrated that studied populations are more differentiated within than among stands, which were grouped into one cluster of genetic similarity. In conclusion, the present distribution of genetically related populations of Scots pine in North-eastern Poland seems to reflect the historical events such as postglacial colonization of Poland from different European refugia and/or human management carried out in the past.