Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has become a widely used technology for the detection of nucleic acids. In this chapter we introduce its use for the discovery of novel sequence polymorphisms and the identification of known DNA changes. We first provide a brief overview about MALDI-TOF MS analysis of nucleic acids. We then elucidate the concept of base-specific cleavage and its use for the discovery of sequence polymorphisms. We also introduce the use of primer extension assays for the classification of known genomic alterations. Finally, we provide a detailed protocol for the implementation of both methods for practical use in a high-throughput setting.