ABSTRACT Genetic resources of red clover (Trifolium pratense L.) are the basis for the improvement of this important forage legume. The objective of this study was microsatellite characterization of the accessions from the collection of the Institute of Field and Vegetable Crops in Novi Sad, Serbia. Molecular evaluation of 46 red clover genotypes was performed by applying the set of 14 primer pairs of microsatellite markers. These primer pairs amplified a total of 187 alleles, with an average of 13.36 alleles per locus and average polymorphism information content (PIC) value was 0.306. The minimum values of Dice genetic distances based on polymorphism of microsatellite markers were found among genotypes NCPGRU2 and NCPGRU5 (0.311) and the highest values of genetic distances were determined for a couple of genotypes Violeta and BGR2 (0.933). The average genetic distance between all pairs of genotypes amounted 0.587. The results of the principal coordinate analysis (PCoA) were consistent with the results obtained on the basis of cluster analysis, except that the PCoA allocated another four genotypes. There was no relationship between groups of genotypes formed by the use of cluster analyses and PCoA with their geographical origin. Analysis of molecular variance of 46 red clover genotypes by the status and ploidy level was significant, but it also suggested a weak genetic differentiation of groups formed on the basis of those characteristics. Observed groups of genotypes, according to the cluster analyses and PCoA of microsatellite data, could be used in future breeding programs for the selection of germplasm.