The human ribosomal DNA (rDNA) locus encodes a variety of long non-coding RNAs (lncRNAs). Among them, the canonical ribosomal RNAs that are the catalytic components of the ribosomes, as well as regulatory lncRNAs including promoter-associated RNAs (pRNA), stress-induced promoter and pre-rRNA antisense RNAs (PAPAS), and different intergenic spacer derived lncRNA species (IGSRNA). In addition, externally encoded lncRNAs are imported into the nucleolus, which orchestrate the complex regulation of the nucleolar state in normal and stress conditions via a plethora of molecular mechanisms. This review focuses on the triplex and R-loop formation aspects of lncRNAs at the rDNA locus in yeast and human cells. We discuss the protein players that regulate R-loops at rDNA and how their misregulation contributes to DNA damage and disease. Furthermore, we speculate how DNA lesions such as rNMPs or 8-oxo-dG might affect RNA–DNA hybrid formation. The transcription of lncRNA from rDNA has been observed in yeast, plants, flies, worms, mouse and human cells. This evolutionary conservation highlights the importance of lncRNAs in rDNA function and maintenance.