Heteroplasmy is the presence of variable mitochondrial DNA (mtDNA) within the same individual. The dynamics of heteroplasmy allele frequency among tissues of the human body is not well understood. Here, we measured allele frequency at heteroplasmic sites in two to eight hairs from each of 11 humans using next-generation sequencing. We observed a high variance in heteroplasmic allele frequency among separate hairs from the same individual—much higher than that for blood and cheek tissues. Our population genetic modelling estimated the somatic bottleneck during embryonic follicle development of separate hairs to be only 11.06 (95% confidence interval 0.6–34.0) mtDNA segregating units. This bottleneck is much more drastic than somatic bottlenecks for blood and cheek tissues (136 and 458 units, respectively), as well as more drastic than, or comparable to, the germline bottleneck (equal to 25–32 or 7–10 units, depending on the study). We demonstrated that hair undergoes additional genetic drift before and after the divergence of mtDNA lineages of individual hair follicles. Additionally, we showed a positive correlation between donor’s age and variance in heteroplasmy allele frequency in hair. These findings have important implications for forensics and for our understanding of mtDNA dynamics in the human body. This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’.