The selection of red clover (Trifolium pratense L.) populations adapted to extreme environmental conditions is of great importance due to continuing climate change. The plant material analyzed with simple sequence repeat (SSR) markers included two parent populations, P1 (cultivar ‘Reichersberger’) and P3 (cultivar ‘Croatia’) and their reselections, which were created after one cycle of selection under cold temperature conditions. The reselections PS1 and PS3 were produced by intercrossing 38 surviving plants of parent populations P1 and P3, respectively. A total of 48 plants from each cultivar and each reselection were randomly selected for SSR analysis. Sixteen SSR primer pairs were selected, taking into account the presence of loci on all seven pairs of red clover chromosomes. An increase in the average frequency of alleles from the initial populations to the populations after one cycle of selection was observed, followed by a decrease in the number of alleles. Out of a total of 16 loci, the Waples neutrality test revealed significant frequency changes at 12 loci from P1 to PS1 and 9 loci from P3 to PS3 populations. The genetic diversity in the studied populations did not change significantly after selection, leaving enough genetic variability as a prerequisite for the success of future selection.