The ability to analyze multiple polymorphic sites rapidly and accurately is crucial in all areas of genetic analysis. We have developed an approach for the detection of multiple point mutations, using allele-specific, mass-labeled, peptide nucleic acid (PNA) hybridization probes, and direct analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The composite mass spectra produced contain peaks of distinct masses corresponding to each allele present, resulting in a mass spectral "fingerprint" for each DNA sample. The hybridization characteristics of PNA:DNA duplexes were found to be highly dependent on both base content and sequence. Results from the analysis of four polymorphic sites contained in exon 4 of the human tyrosinase gene show that this approach is simple, rapid, and accurate with potential applications in many areas of genetic analysis.