Biologic and antigenic properties are often useful for identifying and differentiating orthopoxviruses (OPV). However, polymerase chain reaction (PCR) amplification, with either restriction cleavage or sequencing of amplicons, has been gaining credibility as a more rapid, specific, sensitive, and often cost-saving technique for research and diagnostic laboratories. This chapter is consolidated using prior research papers from our laboratories with three different methods that should be suitable for the preparation of orthopoxvirus DNA from various sources (e.g., clinical specimens or cell cultures) and four different methods for PCR that should be useful for investigating orthopoxvirus species and strains.