Genetics research has benefited tremendously from the release of the human genome sequence. Subsequent technology has been developed and adapted to accommodate the need for faster, easier throughput of genetic assays. Pyrosequencing is a unique system that allows the analysis of genetic variations including single-nucleotide polymorphisms, indels and short repeats, as well as assessing RNA allelic imbalance, DNA methylation status, and gene copy number. Advances in methodology, including multiplex and universal primer applications, have reduced assay cost and improved throughput. This chapter briefly reviews some of the many applications for Pyrosequencing technology.