After completion of the human genome project, the focus of geneticists has shifted to elucidation of gene function and genetic diversity to understand the mechanisms of complex diseases or variation of patient response in drug treatment. In the past decade, many different genotyping techniques have been described for the detection of single-nucleotide polymorphisms (SNPs) and other common polymorphic variants. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is among the most powerful and widely used genotyping technologies. The method offers great flexibility in assay design and enables highly accurate genotyping at high sample throughput. Different strategies for allele discrimination and quantification have been combined with MALDI (hybridization, ligation, cleavage, and primer extension). Approaches based on primer extension have become the most popular applications. This combination enables rapid and reliable multiplexing of SNPs and other common variants, and makes MALDI-TOF-MS well suited for large-scale studies in fine-mapping and verification of genome-wide scans. In contrast to standard genotyping, more demanding approaches have enabled genotyping of DNA pools, molecular haplotyping or the detection of free circulating DNA for prenatal or cancer diagnostics. In addition, MALDI can also be used in novel applications as DNA methylation analysis, expression profiling, and resequencing. This review gives an introduction to multiplex genotyping by MALDI-MS and will focus on the latest developments of this technology.