Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) constitute important sources of genetic variation which provide insight into disease origins and differences in drug responses. The analysis of such genetic variation relies upon the generation of allele-specific products, typically by enzymatic extension or alternatively by the hybridization of DNA probes. MALDI-TOF mass spectrometry (MS) has been widely used as a read-out tool. We developed a distinct enzyme-free, dynamic chemistry-based method of producing allele-specific products for genotyping. In a blind trial, peptide nucleic acid (PNA) probes and aldehyde-modified nucleobases were employed to genotype twelve cystic fibrosis patients for two mutations (one SNP and one indel) linked to this disease. MALDI-TOF MS reported the resulting allele-specific products. Enzyme-free dynamic chemistry permitted the genotyping of twelve individuals for the ΔF508 (indel) and G551D (SNP) mutations. No false positives or negatives were observed, and analysis could be performed in both singleplex and duplex formats. Dynamic chemistry provides a distinct method of allele discrimination with certain advantages over those reported previously. Although PCR amplification is required, the analysis stage is performed without enzymes, and there is no need for the stringent optimization and control of conditions associated with hybridization-based methods of allele discrimination.