Though CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation (IP), mass spectrometry, and ELISA methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5 % CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells, to independently answer these two questions. We find no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing datasets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally-specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function.