DescriptionSingle-stranded DNA (ssDNA) viruses mutate and evolve at similar rates to RNA viruses, and more rapidly than double-stranded DNA viruses. Unlike RNA viruses, the mechanism underlying these rates is unknown. When unpaired in ssDNA, cytosine is inherently unstable, readily deaminating to uracil. These spontaneous events result in cytosine-to-thymine substitutions, and may explain the high mutation and evolution rates of ssDNA viruses. We examined the codon usage bias of ssDNA bacteriophages and eukaryotic viruses, and found that ssDNA viruses consistently overuse thymine at synonymous sites, regardless of the codon preferences of their hosts. This finding is consistent with a persistent cytosine-to-thymine mutation pressure. We further utilized bottleneck passaging to characterize the mutation spectrum of phiX174, a ssDNA bacteriophage, though we were unable to observe mutational bias. Finally, we treated populations of phiX174 with sodium bisulfite, a cytosine-specific deaminating agent, to induce lethal mutagenesis by elevating the mutation rate of cytosine. We were able to successfully drive these populations to extinction, and lethal mutagenesis is the most likely explanation for these observations.