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5S ribosomal DNAs (rDNAs) are arranged in tandem and are often under-represented in genome assemblies. In the present study, we performed a global and in-depth analysis of the 5S rDNAs in the model insect Tribolium castaneum and its closely related species Tribolium freemani. To accomplish this goal, we used our recently published genome assemblies based on Nanopore and PacBio long-read sequencing. Although these closely related species share the 5S rRNA gene sequence with high homology, they show a different organization of the 5S rDNA locus. Analysis of 5S rDNA arrays in T. castaneum revealed a typical tandemly repeated organization characterized by repeat units consisting of the 121 bp long 5S rRNA gene and the 71 bp long nontranscribed spacer (NTS). In contrast, T. freemani showed a much more complex organization of 5S rDNA arrays characterized by two patterns. The first is based on the association of 5S rRNA gene with arrays of a satellite DNA, representing the NTS sequence of the 5S rDNA genes in T. freemani. The second, more complex type is characterized by a somewhat less frequent occurrence of the 5S rRNA gene and its association with longer satellite DNA arrays that are regularly interrupted by Jockey-like retrotransposons. This organization, in which the ribosomal gene is associated with two completely different repetitive elements such as satellite DNAs and retrotransposons, suggests that the 5S rRNA gene, regardless of its crucial function in the genome, could be a subject of extremely dynamic genomic rearrangements. [ABSTRACT FROM AUTHOR]