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Global mitochondrial DNA (mtDNA) methylation has been recently described in bovine and showed particular signatures in both gametes and embryos. Here, we investigated the distribution of mtDNA methylation through strand‐specific mapping of methylation sites to gain perspective on how epigenetic mechanisms can be involved in mitochondrial function. We demonstrate that in both oocytes and embryos, the frequency of methylation is biased towards the light strand (L‐strand), particularly in the gene bodies and in the region containing the L‐strand promoter (LSP). Methylation is not restricted to CpG nucleotides and is not symmetrical on both strands. This configuration reinforces the hypothesis of a specific epigenetic regulation of mtDNA, which is an important observation for the understanding of how mitochondrial function is regulated.