The integrity of DNA is constantly attacked by a wide array of exogenous and endogenous mutagens, one of the most ubiquitous being UV exposure from the sun. UV induces DNA damage, primarily cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts (6-4PPs), that inhibit transcription and replication and can cause mutations. Although UV mutagenesis has been extensively studied, there have been several conflicting reports regarding the impact of cytosine methylation (i.e., 5-methylcytosine) on the induction of UV damage. We hypothesized that the presence of cytosine methylation will promote the formation of CPDs following UVB irradiation in naked yeast genomic DNA. To test this, we examined the impact of cytosine methylation on UV damage formation using a genome-wide and single-nucleotide resolution mapping method known as CPD-seq developed in our lab. CPD-seq was used to map CPDs in UV-irradiated yeast genomic DNA in the presence and absence of cytosine methylation at CpG sites by purified M. SssI CpG methyltransferase. This analysis revealed an average ~2-fold induction of CPD formation in methylated genomic DNA following UVB irradiation as compared to non-methylated controls. We also identified that the magnitude of this induction was dependent on the flanking DNA sequence context. Sequence contexts with a higher propensity to form CPDs in the absence of cytosine methylation showed a weaker CPD induction following methylation. These findings reveal the effects of cytosine methylation on UV damage formation across a eukaryotic genome.