Abstract <p>Primers for amplification of six minisatellite and one microsatellite loci were developed based on the mitochondrial genome of Norway spruce. Their testing on six populations of Siberian spruce and the northern cluster of Norway spruce (120 trees) revealed from three to seven alleles. The multilocus combination of six minisatellites yielded a total of 27 haplotypes. The populations had from two to nine haplotypes, but almost no common ones, and were divided (<i>R</i>_CT = 0.77) into three groups—European Russia, the Urals and the Siberian spruce population (Altai). In a population located at the alpine tree line in the Southern Urals, the haplotypes were not located randomly, which demonstrates the possibility of using the developed markers to identify groups of maternally related trees and study the process of tree colonization of the open areas. In the population from the southern cluster of Norway spruce (Carpathians), 24 trees, only one of the markers had stable amplification and was variable (6 alleles).</p>