The mitochondrial (mt) genome is present in several copies in human cells, and intra-individual variation in mtDNA sequences is known as heteroplasmy. A recent study found that heteroplasmies were highly tissue-specific, site-specific, and allele-specific, suggesting that positive selection is acting on such heteroplasmies; however the functional implications have not been explored. This study investigates variation in mtDNA copy numbers (mtCN) in 12 different tissues obtained at autopsy from 152 individuals (ranging in age from 3 days to 96 years). Three different methods to estimate mtCN were compared: shotgun sequencing, capture-enriched sequencing and droplet digital PCR (ddPCR). The highest precision in mtCN estimation was achieved using shotgun sequencing data. However, capture-enrichment data provide reliable estimates of relative (albeit not absolute) mtCNs. Comparisons of mtCN from different tissues of the same individual revealed that mtCNs in different tissues are, with few exceptions, uncorrelated. Hence, each tissue of an individual seems to regulate mtCN in a tissue-related rather than an individual-dependent manner. Skeletal muscle (SM) samples showed an age-related decrease in mtCN that was especially pronounced in males, while there was an age-related increase in mtCN for liver (LIV) samples. MtCN in SM samples was significantly negatively correlated with both the total number of heteroplasmic sites and with minor allele frequency (MAF) at two heteroplasmic sites, 408 and 16327. Heteroplasmies at both sites are highly specific for SM, occur in more than 40 % of the individuals older than 50 years (with MAF up to 28.2 %), and are part of functional elements that regulate mtDNA replication. We hypothesize that positive selection acting on these heteroplasmic sites is reducing mtCN in SM of older individuals.