Retrotransposons are present in essentially all eukaryotic genomes and come in two basic flavors: those that are bracketed by long terminal repeats (LTRs) and share a common ancestor with retroviruses, and non-LTR retrotransposons that have a distinct lineage and remain transpositionally active in humans. Both types of retrotransposons replicate through an RNA intermediate, stably integrate into the host genome and have accumulated to a very high copy number in mammals and certain plant species. Autonomous elements produce transcripts capable of undergoing reverse transcription, and minimally encode proteins with reverse transcriptase, integrase/endonucleolytic, and nucleic acid chaperone activities. Retrotransposons are currently distinguished from viruses, since the process of retrotransposition is not infectious. However, this boundary may prove to be provisional as we learn more about these mobile genetic elements. The goal of this Special Issue of Viruses is to highlight progress in understanding the mechanism and consequences of retrotransposon movement. Several active research areas may be covered in reviews and research articles, including the roles of cellular modulators and defense systems, retrotransposon expression and replication, retrotransposon-induced mutations and their association with human diseases, and how these widely disseminated elements mold eukaryotic genomes.