A dozen of murine Pol III-derived transcripts, such as tRNAs, 5S ribosomal RNA and repetitive elements, have been identified and play crucial roles in transcription, translation or contribute to structural rearrangements. Pol III transcription is exceptional and differs from Pol II transcription. Unlike Pol II, Pol III does not rely on regulatory control sequences in promoter regions to initiate gene transcription but on gene internal sequences. These intrinsic sequences are recognized by specific regulatory factors and have distinguished three types of Pol III transcripts. Once gene transcription is initiated, Pol III gives rise to transcripts that are usually shorter than 300 nucleotides. Importantly, most Pol III-derived transcripts are part of multi-copy gene families, making it difficult to identify the genomic regions from where the transcripts arrive by using RNA-based methods since sequence reads will map to multiple locations. In contrast, profiling Pol III binding to the genome allows accurate identification of actively transcribed genes since Pol III will not only recognise the repetitive sequence of the gene body but also the unique regions flanking an actively transcribed gene locus. Methods exploring this feature have shown that only a fraction of annotated Pol III genes is actively transcribed in healthy tissue and liver cancer cells.