Methylation of cytosine at the 5-position is a common epigenetic modification in mammalian DNA and plays an important role in regulating gene expression. Oxidized derivatives of 5-methylcytosine were discovered recently, and some of these oxidized derivatives, in addition to being intermediates in an active demethylation pathway, might also function as epigenetic modifications. Oxidized derivatives of thymine are known to be products of DNA damage, although evidence exists that the oxidized thymine derivative 5-hydroxymethyluracil might have an epigenetic role. There is a pressing need to learn more about these modifications, as epigenetic modifications have roles in development and in diseases, including cancer. This emerging area of research requires highly accurate and sequence-specific methods for the detection of cytosine and thymine modifications in DNA. In this Review, we introduce the biochemistry of cytosine and thymine modifications and discuss established detection methods, such as bisulfite sequencing and its modifications, as well as newer methods, which have been developed to overcome the substantial obstacles associated with studying these modifications in genomic DNA.