Human pathogenic orthopoxvirus (OPV) including variola, monkeypox, vaccinia and cowpox is an important viral genus for human infection. Sharing highly similar viral protein sequences, the genus OPV poses diffi culty to diagnosis using serological tests. Thus, molecular detection is an important method for quick detection and identifi cation of orthopoxvirus infection. In this study, two novel targeting regions of VAR1 and VAR2 located on late 16-Kda putative membrane protein (LPMP) and 39-kDa core protein (CP) genes have been identifi ed for developing PCR assays for OPV. SYBR green- and probe-based real-time PCR assays have been established for detecting and/or typing DNA of OPV. Two pan-OPV SYBR green-based PCR assays targeting LPMP and CP genes show high sensitivity in detecting pan-OPV DNAs in levels of 1 copy DNA/reaction or 10 pfu/ml. A pan-OPV probe-based real-time PCR assay targeting on LPMP gene also exhibits high sensitivity in detecting pan-OPV DNAs in levels of 1 copy DNA/reaction or 10 pfu/ml. Monkeypox- and smallpoxspecifi c probe-based real-time PCR assays targeting CP genes can sensitively and accurately detect both important DNAs of human OPV, namely monkeypox and variola in the level of 10 copies and 1 copy DNA per reaction, respectively. This study provides alternatives for PCR detection of orthopoxvirus, monkeypox and smallpox DNA, which would be valuable for clinical diagnosis of human pathogenic orthopoxvirus infections and route laboratories.