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Mitochondria are cellular organelles regulating metabolism and cell death pathways. This study examined changes in mitochondrial membrane potential (ΔΨm) throughout the stages of preimplantation development in mouse embryos conceived either in vivo or in vitro and human embryos donated to research from IVF. Embryos stained with the ΔΨm‐sensitive dye (JC‐1) were quantified for the ratio of high‐ to low‐polarized mitochondria using a deconvolution microscope. Overall, mouse zygotes and early embryos contain a subset of high‐polarized mitochondria with a progressive increase in the ratio of ΔΨm observed with increasing cleavage. A transient increase in the ratio of high to low ΔΨm was observed in in vivo fertilized 2‐cell stage embryos, coincident with embryonic genome activation in the mouse, but not in 2‐cell embryos obtained through IVF. We further observed that arrested mouse 2‐cell embryos …