With the discovery of several matrices which enable the ionization of DNA and RNA, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a powerful platform for the study of nucleic acid sequence changes (e.g., mutations, single nucleotide polymorphisms (SNPs), insertion/deletion, alternative splicing, etc.), amount changes (e.g., copy number variation, gene expression, allele expression, etc.), as well as modifications (e.g., methylation of genomic DNA, post transcriptional modification of tRNAs and rRNAs). Two major strategies have been employed to characterize these changes. Primer extension reactions are designed for genotyping of known polymorphic sites and determining the levels of gene or allele expressions. Base-specific cleavage reactions are used for discovery of unknown polymorphisms and characterization of modifications. These two assays usually generate nucleic acid fragments less than 30 bases in length, which is the ideal mass range for MALDI-MS. Here we review the basic concepts of these assays, sample analysis techniques, and their applications published in recent years.