Background <p>Ancient permafrost, formed during past glacial periods, is widespread in Siberia and other Arctic regions. Even though these soils have remained below 0 °C over geological time periods, the widespread presence of bacteria and archaea in permafrost is well documented. However, the diversity of viruses in ancient permafrost of different geological ages and their potential ecological roles are still poorly understood.</p> Results <p>We applied metagenomics to characterize viruses from Middle to Late Pleistocene permafrost sediments from Siberia. A total of 2697 viral operational taxonomic units (vOTUs) were recovered through metagenomic assembly and virus identification. Viral diversity in the Middle Pleistocene permafrost was much higher than that in the Late Pleistocene. The virus communities at different depths of the Middle Pleistocene permafrost showed great similarity to each other but were significantly different from those of the Late Pleistocene, with <i>Azeredovirinae</i>, <i>Peduoviridae</i>, and <i>Fiersviridae</i> being the predominant viruses in Pleistocene permafrost. The viruses were predicted to carry auxiliary metabolic genes potentially involved in cold adaptation and elemental (C and N) biogeochemical cycling in ancient permafrost. The virus-host prediction revealed that microorganisms such as bacteria and archaea are the main hosts and only a few eukaryotic hosts were identified.</p> Conclusions <p>Our results show that permafrost viral communities in Siberia exhibit remarkable diversity and may have played a significant ecological role in ancient permafrost over geological time. While the release of viruses from thawing deep permafrost could pose a relatively small risk to human health and environment, it could have significant impacts on the carbon cycle, potentially influencing climate change feedback in permafrost regions.</p>