Previous work has examined the use of mitochondrial DNA (mtDNA) damage as a biomarker of cumulative sun exposure in human skin. These studies have simply compared mtDNA damage between sun-protected and sun exposed skin. This approach is limited because non-melanoma skin cancer (NMSC) is predominantly formed on body sites which are 'usually' sun exposed as opposed to sites which are 'occasionally' sun exposed and as such they differ in their cumulative ultraviolet (UV) exposure. This study addresses this limitation by investigating the frequency of occurrence of a rarely reported 3895 bp mtDNA deletion in 104 age-matched human skin samples taken from different sun-exposed body sites. There was a significant increase in the deletion frequency with increasing UV exposure (p<0.0001). Furthermore there was a significantly greater deletion frequency in 'usually' sun exposed compared with 'occasionally' sun-exposed body sites in both the dermis (p=0.0018) and epidermis (p<0.0001). Investigation of the 3895 bp deletion in the same NMSC samples used in a previous study of the 4977 bp common deletion, showed a greater frequency of the 3895 bp deletion (8/10 vs 4/10, respectively). Additionally, we have linked the 3895 bp deletion with the UVR component of sunlight by inducing the deletion in vitro with repetitive sub-lethal doses of a UVA+UVB light source.