Since the early 2000s, Pyrosequencing(®) technology has been adapted for various instrument platforms to enable users to examine the role of epigenetic DNA methylation in gene expression regulation, genetic markers for specific phenotypes in livestock, drug resistance development in pathogens, and polymorphisms in forensic samples of mitochondrial DNA.The instruments, software, and chemistry have been modified to facilitate different sample throughputs and sample amounts. Just recently, major changes have been implemented to enable increased read length and more precise Pyrosequencing results. These improvements were made possible through a number of changes to various system components. In addition, assay development has been streamlined through the availability of optimized PCR and Pyrosequencing reagents, automated assay design tools, and a number of predesigned Pyrosequencing assays.In future, instruments with smaller footprints and the ability to automate crucial steps of the Pyrosequencing protocol will be available and will provide even more convenient and standardized Pyrosequencing analysis with flexible throughput.