The increased presence of 5-methycytosine at gene promoter regions may be diagnostic of cancer. However, there are many stages in the measurement of gene promoter 5-methylcytosine content where inaccuracies may occur, and this may prevent the use of these measurements for diagnostic or prognostic purposes. A high accuracy LC-MS system was developed for measuring the degree of methylation in two 100 base pair amplicons generated by the polymerase chain reaction (PCR) and in which 5-methylcytidine had been synthetically incorporated. Nucleotide monophosphate reference materials were used to calibrate the peak area ratio of cytidine and 5-methylcytidine to their mole ratio in enzymatic hydrolysates of the amplicons, thus enabling metrological traceability of the methylation ratio to the mole. The methylation values obtained agreed closely with the reference values assigned to the materials. A measurement uncertainty budget was completed and showed that the moisture content of the nucleotide monophosphate reference materials was the largest source of uncertainty in the methylation ratio measurement. Measurement of an oligonucleotide supplied with the materials provided evidence that such materials may be used for calibration of DNA methylation ratios without the need for measurement of moisture content. This raises the possibility that submicrogram amounts of appropriately characterized oligonucleotide reference materials could be used to calibrate methylation ratios obtained by contemporary methodologies (such as PCR after bisulfite conversion of genomic DNA) yielding values that are traceable to the International System of Units (SI). Such calibrated gene methylation measurements would then be internationally comparable as required for effective diagnostic and prognostic measurements.