Mitochondrial DNA content remains constant between the mature egg and the blastocyst stage in mammals, making this the only period in development when genotypes segregate to daughter cells without the confounding effect of genotype replication. To analyze the segregation patterns of mitochondrial DNA during preimplantation development, we introduced polymorphic mitochondria either peripherally (cytoplast transplantation) or in the perinuclear vicinity (karyplast transplantation) into zygotes. Genotype ratios were significantly more variable among blastomeres from cytoplast (coefficient of variation = 83.8%) than karyoplast (coefficient of variation = 34.7%) reconstructed zygotes. These results suggest that heteroplasmy caused by polymorphic mitochondria positioned in the periphery of oocytes at the time of fertilization shows a more stringent segregation pattern than when the organelle is in the vicinity of the nucleus. Moreover, donor-to-host mitochondrial genotype ratios in karyoplast-derived groups increased significantly during development, particularly in the C57BL/6 group, where the ratio practically doubled between the four-cell (17.3%) and the blastocyst stage (29.6%). Although the mechanisms controlling this preferential replication of nuclear-type mitochondrial DNA are unknown, it is suggested that access to nuclear-derived transcription and replication factors could lead to the preferential replication of perinuclear mitochondrial genotypes during morula and blastocyst formation.