Abstract Background Mitochondria have their own circular multi-copy genome (mtDNA), and abnormalities in the copy number are implicated in mitochondrial dysfunction, which contributes to a variety of aging-related pathologies. However, not much is known about the genetic correlation of mtDNA copy number across multiple generations and its physiological significance. Methods We measured the mtDNA copy number in cord blood or peripheral blood from 149 three-generation families, specifically the newborns, parents, and grandparents, of 149 families, totaling 1041 individuals. All of the biological specimens and information were provided by the Tohoku Medical Megabank Project in Japan. We also analyzed their maternal factors during pregnancy and neonatal outcomes. Results While the maternal peripheral blood mtDNA copy number was lower than that of other adult family members, it was negatively correlated with cord blood mtDNA copy number in male infants. Also, cord blood mtDNA copy numbers were negatively correlated with perinatal outcomes, such as gestation age, birth weight, and umbilical cord length, for both male and female neonates. Furthermore, the mtDNA copy number in the infants born to mothers who took folic acid supplements during pregnancy would be lower than in the infants born to mothers who did not take them. Conclusions This data-driven study offers the most comprehensive view to date on the genetic and physiological significance of mtDNA copy number in cord blood or peripheral blood taken from three generations, totaling more than 1000 individuals. Our findings indicate that mtDNA copy number would be one of the transgenerational biomarkers for assessing perinatal outcomes, as well as that appropriate medical interventions could improve the outcomes via quantitative changes in mtDNA.