Various studies have demonstrated that mitochondrial DNA (mtDNA) heteroplasmy tends to increase with age and that the observed frequency of heteroplasmy among populations mostly depends on the way it is measured. Therefore, we investigated age-related association on the presence of mtDNA heteroplasmy within the hypervariable segment 1 (HVS-I) in a selected study group. The study group consisted of 300 maternally unrelated Latvians ranging in age from 18 to over 90. years. To determine the optimal method for mtDNA heteroplasmy detection, three approaches were used: (i) SURVEYOR Mutation Detection Kit, (ii) sequencing and (iii) denaturing gradient-gel electrophoresis (DGGE). Among the studied individuals, 30.3% were found to be heteroplasmic. The distribution of heteroplasmy statistically significantly increased with individuals’ age (17%; 95% confidence interval [CI] 0.095-0.244 in the 18-40. year age group vs. 39%; [CI] 0.294-0.487 in the textgreater 90 year age group). Heteroplasmy occurred in a total of 21 different positions within HVS-I, and was the most frequent at fast-mutated positions 16189, 16304 and 16311. The results indicate that heteroplasmy in HVS-I is relatively common and occurs in a broad spectrum of sites. The above is supported by evidence to eventual increase of the probability of heteroplasmy with age due to specific mitochondrial haplogroup background. © 2011 Elsevier Inc.