Aim. The region encoding 5S rRNA (5S rDNA) is present in the genome of all eukaryotic organisms. The 5S rDNA represents a universal model for studying the molecular evolution of the tandemly arranged repeated sequences. However, in the family Solanaceae, the molecular organization of 5S rDNA has been investigated only for few genera. In this regard, we decided to characterize the molecular structure of the 5S rDNA of Mandragora autumnalis, a representative of the Mandragoreae tribe, which occupies an isolated position in the nightshade family. Methods. PCR amplification, cloning and sequencing. Results. Two 5S rDNA clones of M. autumnalis were sequenced. It was found that in the genome of this species only one class of the 5S rDNA repeats possessing the 103 bp-long intergenic spacer (IGS) is present. This is the smallest size of IGS known to date for the Solanaceae family. The 5S rDNA IGS of M. autumnalis demonstrates a moderate level of sequence-similarity with the IGS of other representatives of the subfamily Solanoideae. Conclusions. The results support the current opinion about the isolated taxonomic position of the genus Mandragora within the subfamily Solanoideae. The increased level of similarity was found in the IGS regions, which contain potential external elements of the RNA polymerase III promoter and terminator. The mutations occurred in the part of the IGS up-stream of the coding region had a compensatory nature, which ensured that the external elements of the promoter were preserved during the evolution. Keywords: 5S rDNA, intergeneric spacer, molecular evolution, Mandragora autumnalis, Solanaceae.