Until recently, it was believed that the genomes of higher organisms contain, in addition to the four canonical DNA bases, only 5-methyl-dC (m⁵dC) as a modified base to control epigenetic processes. In recent years, this view has changed dramatically with the discovery of 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNA from stem cells and brain tissue. N⁶-methyldeoxyadenosine (m⁶dA) is the most recent base reported to be present in the genome of various eukaryotic organisms. This base, together with N⁴-methyldeoxycytidine (m⁴dC), was first reported to be a component of bacterial genomes. In this work, we investigated the levels and distribution of these potentially epigenetically relevant DNA bases by using a novel ultrasensitive UHPLC–MS method. We further report quantitative data for m⁵dC, hmdC, fdC, and cadC, but we were unable to detect either m⁴dC or m⁶dA in DNA isolated from mouse embryonic stem cells or brain and liver tissue, which calls into question their epigenetic relevance.