A method for one-stage rapid identification of four orthopoxvirus species pathogenic to humans based on multiplex polymerase chain reaction (MPCR) was developed. Five pairs of oligonucleotide primers--one, genus-specific; and the rest, species-specific for variola, monkeypox, cowpox, and vaccinia viruses, respectively--were used concurrently for MPCR assay of orthopoxvirus DNAs. Specificity and sensitivity of the method developed were evaluated using DNAs of 57 orthopoxvirus strains, including the DNAs isolated from human case clinical materials.