During the transition period most high-yielding dairy cows suffer from negative energy balance due to decreased energy intake and increased energy demands required for milk synthesis. Mitochondria are the main sites of energy metabolism in mammalian cells and their number varies depending on age, sex, organ, and physiological or pathological conditions. Mitochondria exhibit their own genome, the mitochondrial DNA (mtDNA), and its copy numbers reflect the abundance of mitochondria within a cell. In the course of lactation, environmental, physiological, and energetic conditions alter. We hypothesized that these changes may influence the number of mtDNA/cell in dairy cows and thus investigated the number of mtDNA copies in blood during early and late lactation. German Holstein cows (n = 21; BCS: 3.0 ± 0.1) were fed according to their requirements. Estimated total energy requirements were calculated by adding the requirements for maintenance and milk production. Blood samples from the jugular vein were collected 3 and 35 wk postpartum. Genomic DNA was extracted from whole blood using a commercially available kit. Based on the amplification of the 12S rRNA (mtDNA target gene) and the β-globin (reference gene) to normalize the DNA content in each sample, mtDNA copy number was assessed by a multiplex qPCR. Data (mean ± SEM) were analyzed by the pairwise Student’s t-test (SPSS 22). In early lactation the number of mtDNA (87.4 ± 15.5 copies/cell) was about twice as much as in late lactation (46.0 ± 3.7; P = 0.008). In early lactation energy demands are increased compared with late lactation, as indicated by the estimated total energy requirements for wk 3 (132 ± 8.26 MJ NE_L) and wk 35 (103 ± 5.77 MJ NE_L). The greater energy demands in early lactation were accompanied by elevated mtDNA copy numbers in peripheral blood when compared with late lactation. The observed changes meet the expectation that the metabolic load during early lactation requires more mitochondria. Peripheral blood thus forms a suitable matrix to assess the cellular content of mitochondria and the cellular energetic status of dairy cows when tissues are not accessible.