Smallpox results from infection by variola virus (genus Orthopoxvirus, family Poxviridae). At least two strains of virus exist: the more virulent strain causes variola major and the less virulent strain causes variola minor.

The Orthopoxvirus genus also includes the vaccinia, monkeypox, cowpox, camelpox and ectromelia viruses; these agents resemble each other in electron micrographs and in many culture systems. They are also serologically cross–reactive. Monkeypox, which usually found in Africa, closely resembles smallpox; in monkeypox infections, the mortality rate is lower and the cervical and inguinal lymph nodes are highly enlarged.