With the onset of lactation, energy requirements rapidly increase in high-yielding dairy cows. To adapt to lactation, energy metabolism needs to be regulated and coordinated among the key organs, namely adipose tissue (AT), liver, and mammary gland. Mitochondria are the main site for energy production in mammalian cells and their number depends on the energy demand and physiological state of each individual. Mitochondria have their own DNA and therefore the abundance of mitochondria in a cell is reflected by the copy number of mitochondrial DNA (mtDNA). Age-related differences of mtDNA are known for mice and humans, in which mtDNA varies between different tissues. However, little is known about mtDNA copy numbers in dairy cows; we thus aimed to provide an overview of mtDNA copy numbers in liver, subcutaneous (sc) AT and mammary gland of lactating dairy cows. Lactating German Holstein cows (n = 21; BCS: 3.0 ± 0.1) were fed according to their requirements. Liver, mammary gland and subcutaneous (sc) AT from the tailhead region were sampled during early lactation (3 wks postpartum), in which the estimated total energy requirement was 132 ± 8.26 MJ NE_L. Biopsies were immediately snap frozen after sampling. Genomic DNA was extracted using commercially available kits and the number of mtDNA copies/cell was quantified by a multiplex qPCR, targeting the 12S rRNA gene and using b-globin as reference gene. Tissue-specific differences were examined by Student’s t-test (SPSS 22). Data are presented as means ± SEM. The number of mtDNA copies/cell in liver (360 ± 22.3 copies/cell) was 7.7-fold and 5.3-fold higher (P < 0.001) than in scAT (46.7 ± 2.32 copies/cell) and mammary gland (68.3 ± 4.66), respectively. Moreover, mammary gland contained 1.5-fold more mtDNA copies/cell than scAT (P = 0.001). The differences in mtDNA content observed between the organs investigated herein are presumably reflecting their metabolic activity during the first weeks of lactation with liver playing a key role.