Background Mitochondrial DNA (mtDNA) haplogroups (hgs) are evolutionarily conserved sets of mtDNA SNPs. Associations of hgs with geographical origin, disease and physiological characteristics have been reported, but have frequently not been reproducible. We assessed, using 418 mtDNA SNPs on the PsychChip (Illumina), the spatio-temporal distribution of mtDNA hgs in DNA isolated from geographically un-biased dried blood spots (DBS), collected from 1981 to 2005 through the Danish National Neonatal Screening program. Methods As part of the iPSYCH (www.iPSYCH.au.dk) recruitment protocol, 24,651 singletons (47.1% female), born between May 1 1981 and Dec 31 2005 were selected at random from the Danish Central Person Registry. The Samples were extracted from the Danish National Biobank and Genotyped at the Broad Institute as part of iPSYCH and PGC. Haplotyping of mtDNA was performed using the defining SNPs reported in www.phylotree.org. First the affiliation to macro-hg i.e. L0 – L6, M, N, R, and then to hgs – units more distal in the cladogram was established. Ancestry estimation was done using ADMIXTURE 1.3.052. Briefly, a reference population consisting of Human Genome Diversity Project (HGDP) ( http://www.hagsc.org/hgdp/ ) genotyping SNP data set, supplemented with representative samples of danes (716 individuals) and greenlanders (592 individuals) available at SSI, was used. Results The hg distribution was typically Northern European, and hgs were highly variable based on median-joining analysis, suggesting multiple founder events. Considerable heterogeneity and variation in autosomal geno-geographic affinity (ancestry) was observed. Thus, individuals with hg H exhibited 95%, and U hgs 38.2% - 92.5%, Danish ancestry. Significant clines between geographical regions and rural and metropolitan populations were found. Over 25 years, macro-hg L increased from 0.2% to 1.2% (p=1.1⁎E-10), and M from 1% to 2.4% (p=3.7⁎E-8). Hg U increased among the R macro-hg from 14.1% to 16.5% (p=1.9⁎E-3). Geno-geographic affinity, geographical skewedness, and sub-hg distribution suggested that the L, M and U increases are due to immigration. Discussion This study shows that the distribution of mtDNA hgs in Denmark is highly dynamic and complex. It comprises 1.6% of the Danish population over a 25-year period, and is by far the largest ever performed of the distribution of mtDNA hgs in any country. The method of collecting stored DBS from the PKU biobank, where the coverage is ~99%, enabled us to survey a true population based sample, where the time and place of birth was known from national electronic registries. This is in contrast to most other population genetic studies of adults sampled in a specific bias-prone context, e.g. hospitalized patients or localized samplings. The mtDNA haplotyping was based on array data with only 418 mtDNA SNPs and as a consequence not all sub-hgs could be called. The stringent adherence to specific SNPs left a number of persons, normally in the order of 1–2%, not haplotypable. An advantage of only using a limited set of markers is that confounding due to private variants is avoided.