Pyrosequencing� technique has been widely used to perform both single-nucleotide polymorphism detection and quantitative DNA methylation analysis. Simplex Pyrosequencing is sufficient to interrogate more than one polymorphic site if these gene variants are within the reach of the sequencing reaction. For polymorphisms far apart from each other or located on different genes, multiple simplex analyses are required. To reduce the number of simplex reactions, multiplex Pyrosequencing becomes a useful alternative method. The multiplex reaction is performed in the presence of single or multiple templates with several sequencing primers. Factors such as primer selection for the PCR and Pyrosequencing reaction, generation of optimal nucleotide dispensation order, use of internal and external controls, preparation of instrumentation, and Pyrogram� interpretation are essential to the success of the multiplexing. In this chapter, the mouse 45S rRNA gene is used to present two general multiplex Pyrosequencing protocols for determining DNA methylation and allele frequency in the spacer promoter region of this gene.