Abstract Tandemly arranged repetitive regions (repeats) that encode 5S rRNA (5S rDNA) are an indispensable component of eukaryotic genomes. Typically, 5S rDNA repeats within a genome are very similar due to the concerted nature of the evolution of this type of repeats. Each 5S rDNA repeat consists of an evolutionarily conserved coding sequence (CDS) and a variable intergenic spacer (IGS). 5S rDNA is a popular model for studying the molecular evolution of repetitive sequences, and the high rate of IGS mutations determines its wide use in phylogenetic analysis of closely related taxa. Nevertheless, 5S rDNA remains unexplored for many groups of higher plants, especially for the Plumbaginaceae family. Some taxa of this family are endemic to southern Ukraine and listed in the Red Book. However, their taxonomic status is controversial, and its clarification requires the use of molecular phylogenetic methods. In this work, we examined the molecular organization of 5S rDNA for representatives of four genera of the tribe Limonieae, the largest in the family Plumbaginaceae. It was shown that the CDS of 5S rDNA of representatives of the genera Limonium, Armeria, and Ceratolimon possess single mutations that do not affect the formation of the secondary structure of 5S rRNA. In contrast, in the genomes of Goniolimon species, in addition to functionally normal 5S rDNA repeats, numerous pseudogenes were found that do not evolve in a concerted manner and contain numerous mutations in the CDS that disrupt the secondary structure of 5S rRNA. A significant phylogenetic distance between representatives of the subgenera Pteroclados and Limonium of the genus Limonium indicates that Pteroclados can be considered a separate genus. The high rate of molecular evolution makes 5S rDNA IGS a convenient tool for the reconstruction of phylogenetic relationships within the studied genera of the tribe Limonieae and the barcoding of Ukrainian endemics of the genus Limonium.