Smallpox, caused by the Variola major virus, is considered to be one of the most lethal of all potential biological weapons and has far-reaching consequences. Real-time polymerase chain reaction (PCR) assays are available as a reliable diagnostic tool to detect members of the genus Orthopoxvirus. In addition real-time PCR assays specific for the variola virus have been developed that distinguish it from other orthopoxviruses. However, a positive identification of variola spp. does not classify the virus as the one that causes smallpox (V. major) or as the variant (Variola minor) that causes a much less severe form of the disease. This study reports the development of a real-time PCR minor groove binder (MGB)-Eclipse probe assay utilizing a sequence within the variola B9R/B10R gene complex that reliably differentiates V. major from V. minor by specific probe melting temperatures (T(m)s) and genotyping analysis. The MGB-Eclipse probe assay is an important step beyond the standard TaqMan-MGB assay and we feel this is a significant addition to our current variola species identification algorithm with TaqMan-MGB assays that target the B9R and B10R genes. The probe T(m)s for V. major and V. minor were 62.71 (+/-0.05) and 53.97 (+/-0.44) degrees C, respectively (P=<0.001). We also used the identical sequence to develop a TaqMan((R))-MGB assay that specifically detected V. minor but not V. major variants by qualitative analysis.