Eukaryotic and prokaryotic genomes are replicated with amazingly high fidelity to assure faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and excise mistakenly incorporated nucleotides using their intrinsic exonucleases. Cell also possess a variety of specialized DNA polymerases that help to overcome replication blocks when occasional unrepaired DNA lesions stall the replication machinery. The translesion synthesis (TLS) polymerases have an extremely low fidelity during copying undamaged DNA substrates, such that uncontrolled participation of these polymerases in DNA replication could present a threat to the genome stability. In this article, we discuss the properties of prokaryotic and eukaryotic TLS polymerases and their roles in modulating the rate of spontaneous and genotoxicant-induced mutations. We also review recent insights into the molecular mechanisms regulating the participation of error-prone TLS polymerases in the genome replication. Finally, we discuss the relationship between the functions of TLS polymerases and human disease.