The use of mtDNA damage as a biomarker of cumulative sunlight exposure in human skin is a relatively new field of research. Previous investigations have simply compared the frequency of occurrence of the mtDNA common deletion (CD), and to a much lesser extent that of tandem duplications (TDs), to distinguish between sun-protected and sun-exposed skin. This approach is limited because non-melanoma skin cancer is predominantly formed on body sites that are "usually" sun-exposed as opposed to sites that are "occasionally" sun-exposed and as such they differ in their cumulative UV exposure. This study addresses this limitation by investigating the frequency of occurrence of the CD and TDs in 116 age-matched human skin samples taken from three different sun-exposed body sites. There was a greater frequency of the mtDNA damage in "usually" sun-exposed compared to "occasionally" sun-exposed body sites for both the CD and the TDs (P < 0.0001 and P = 0.058, respectively). In addition, we identified a 260 bp triplication of the mtDNA D-loop for the first time in skin. No evidence of the CD or TDs was observed in sun-protected (ie rarely exposed) skin (n = 20). Comparatively little is known about mtDNA damage in prolonged skin cell culture. We have furthered this work by studying the level of the CD and the frequency of the TDs during continued culture of human fibroblasts derived from skin samples taken from usually sun-exposed sites (n = 7 patients). The level of the CD decreases with culture, whereas the frequency of TDs can be maintained.