Background: Single nucleotide polymorphisms (SNPs) of mitochondrial DNA (mtDNA) are involved in physiological and pathological conditions. We developed a rapid, accurate, highly sensitive and high-throughput approach with low cost to identify mtDNA SNPs. Methods: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect 18 SNPs of mtDNA by uniplex and multiplex assays. The sensitivity and specificity of the MALDI-TOF MS were evaluated. The accuracy of the approach was validated by the comparison of using the robust sequencing analysis. Results: The detection limit achieved with the assays corresponded to the identification of five-genome equivalence of mtDNA per reaction after first round PCR amplification. The testing system enabled the discrimination of as little as 5% of mtDNA polymorphism in the predominating background of mtDNA not containing the SNP. No false positive and false negative results were obtained using the uniplex and multiplex MALDI-TOF MS assays for the analysis of the 18 SNPs compared with those obtained by sequencing analysis. Conclusions: Possible fields which could benefit from this powerful and sensitive tool include forensic medicine, tracing of matrilineage, transplantation immu-
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