After completion of the human genome sequence the search for differences among individual genomes has become the centre of focus for geneticists. Two different types of polymorphism-single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs)-are major sources of genetic diversity and are of widespread use in genetic analysis. A plethora of genotyping techniques have been developed, and mass spectrometry (MS) is among the most widely used analytical platforms. The most striking advantage of mass spectrometric genotyping assays over others is the use of the measured molecular mass information for allele calling. The molecular mass is less error-prone than other sequence-specific parameters, including migration times, retention times, or hybridization yields, as it represents an intrinsic property of a nucleic acid molecule that is directly related to its nucleotide composition. Mass spectrometric assays can roughly be divided into two major groups-matrix-assisted laser desorption/ionization (MALDI)-based and electrospray ionization (ESI)-based assays. An important subdivision of ESI-based genotyping methods are approaches that originate from the hyphenation of liquid chromatography (LC) to MS. The principles of these three classes of mass spectrometric genotyping techniques are summarized in this review. Possibilities and limitations are critically discussed to assist scientists, especially non-experts in MS, in choosing the appropriate mass spectrometric assay for genotyping a genetic marker of interest. [figure: see text]