Orthopoxvirus species were identified and differentiated by polymerase chain reaction amplification of genome DNA using a single primer-pair based on sequences coding for the major protein component of the cowpox virus acidophilic-type inclusion body (ATI). DNA available for 6 of 8 Old World (cowpox, variola, monkeypox, camelpox, ectromelia and vaccinia viruses) and 3 New World (skunkpox, volepox, and raccoonpox) resulted in amplicons that ranged in size from 510 to 1673 base pairs depending on the species, except for raccoonpox virus DNA which did not amplify. XbaI digest gel electrophoresis profiles of the amplicons improved resolution of the differences.