Human genomic and mitochondrial DNA contain large numbers of single-nucleotide polymorphisms (SNPs), many of which are linked to known diseases. Rapid and accurate genetic screening for important SNPs requires a general methodology which is easily implemented. We present here an approach to SNP discrimination based on high-specificity hybridization of peptide nucleic acid (PNA) probes to PCR-amplified DNA. The assay is directly applied to polymorphisms located within hypervariable region 1 of the human mitochondrial genome and type 1 suballeles of the human leukocyte antigen DQ alpha gene. Captured, single-stranded DNA molecules prepared by PCR amplification are hybridized with PNA probes in an allele-specific fashion. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is then used for rapid, precise, and unambiguous detection and identification of the hybridized PNA probes. Since PNA oligomers bind strongly to complementary DNA under minimal salt conditions, the use of PNA probes is compatible with MALDI-TOFMS. The unparalleled ability of MALDI-TOFMS analysis in terms of molecular weight resolution and accuracy, in conjunction with the highly specific PNA hybridization afforded by this method, offers promise for development into a multiplexed, high-throughput screening technique.