Aim. To study the genetic diversity of tree species it is necessary to use only those regions of genome, which evolve at the highest rate, such as 5S rDNA. To estimate the potential of 5S rDNA to be used as a molecular marker for genogeographic studies, the molecular organization of this genomic region was compared between samples from two geogra-phically remote Bulgarian and Ukrainian populations of Norway maple, Acer platanoides. Methods. PCR amplification, cloning and sequencing. Results. It was shown that in the genome of A. platanoides the 5S rDNA sequences are highly similar. However, in the 5S rDNA intergenic spacer (IGS) of A. platanoides from the Bulgarian population three copies of GTCCGTT subrepeats are present, whereas only one copy of this sequence occurs in plants from the Ukrainian population. Except for different number of subrepeats, the 5S rDNA sequences of the Bulgarian and Ukrainian samples of A. platanoides are identical. In two taxonomically distant species, A. platanoides and A. pseudoplatanus, the region of 5S rDNA IGS, which contains the potential external elements of the promoter of RNA polymerase III, shows no difference, and therefore evolves at a lower rate than other parts of the IGS. Conclusions. The identity of the potential external promoter elements within the genus Acer supports the idea that this part of the IGS could be involved in the initiation of 5S rDNA transcription. The presence of different numbers of sub-repeats in the 5S rDNA IGS in various populations of A. platanoides makes them suitable for identification of intraspecific forms and for evaluation of the intraspecific genetic diversity of A. platanoides. Keywords: 5S rDNA, molecular evolution, sub-repetitions, intraspecific variability, Acer.