The methylation of cytosine to 5-methylcytosine (5-meC) is an important epigenetic DNA modification in many bacteria, plants and mammals, but its relevance for important model organisms including Caenorhabditis elegans and Drosophila melanogaster is still equivocal. By reporting the unambiguous presence of 5-meC in a broad variety of wild, laboratory and industrial yeasts, a recent study also challenged the dogma about the absence of DNA methylation in yeast species. We would like to bring to attention that the high-sensitive gas chromatography/tandem mass spectrometry method employed involved separating cytosine and 5-meC from the deoxypentose backbone through hydrolysis, and is thus unselective about different origins of 5-meC. We employed an alternative LC-MS/MS protocol where by targeting 5-methyldeoxycytidine moieties after enzymatic digestion, only 5-meC specifically derived from DNA is quantified. This technique unambiguously identified cytosine DNA methylation in Arabidopsis thaliana (14.0% of cytosines methylated), Mus musculus (7.6%), and Escherichia coli (2.3%). Despite achieving a detection limit at 250 attomoles (corresponding to <0.00002 methylated cytosines per non-methylated cytosine), we could not confirm any cytosine DNA methylation in laboratory and industrial strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces boulardii, Saccharomyces paradoxus or Pichia pastoris. The protocol however unambiguously confirmed DNA methylation in adult Drosophila melanogaster at a value (0.034%) that is up to two orders of magnitude below the detection limit of bisulphite sequencing. Thus, 5-meC is a rare DNA modification in drosophila, but absent in yeast.