Cytosine methylation is the leading epigenetic modification on DNA playing a role in gene regulation. Methylation can occur in cytosines of any nucleic acids in cytosol (as mitochondrial DNA, mtDNA) and in nuclear DNA (ncDNA). mtDNA exists as multiple copies within numerous mitochondria. This suggests that the number of mitochondria and mtDNA copy number can indicate the presence of a significant amount of DNA methylation within total DNA methylation detected. However, immunofluorescence method does not have a step to discriminate the staining between ncDNA and mtDNA. Antibodies used in immunological methods are methylation-specific but not selective for DNA type and they can bind to methylated cytosines in any DNA within the specimen. Current study aimed to understand whether mtDNA methylation interferes with the detection of nuclear DNA methylation by immunofluorescence and affinity enrichment (ELISA) in different mammalian cells. Experiments were performed to distinguish methylation between mtDNA and ncDNA. Immunofluorescence showed that there was no significant difference in the detected amount of methylation between mitochondrial and nuclear DNA. But ELISA revealed that up to 25% of cellular methylation was derived from mitochondria. This suggests that significant contamination of mtDNA methylation with ncDNA methylation can result in overestimation of the quantitative level of nuclear methylation.