The virus of contagious ecthyma (CEV), also known as orf virus (ORFV) is the etiological agent of contagious ecthyma (CE) in sheep and goat and belongs to the Parapoxvirus genus, family Poxviridae. In some cases, CE can be confused with vesicular diseases so there is need for differentiation especially because, according to the standards of the National Program for the Eradication of FMD (PNEFA), goats and sheep are not vaccinated against Foot and Mouth Disease (FMD), acting as sentinel animals. Although initial studies have demonstrated the usefulness of the polymerase chain reaction (PCR) as a diagnostic test, there are no studies involving its use on Brazilian field samples, which may be genetically distinct from previously studied samples, as described in a study of restriction sites analysis of Brazilian CE samples. This work was conducted with the goal of standardizing a PCR (qPCR) test using SYBR Green I dye for molecular diagnosis of EC in DNA extracted from lesions of affected animal or cell culture inoculated in field samples. The products were detected with qPCR dissociation curve analysis which showed a peak at 88 ºC indicating that positive samples have only one specific amplification product. All DNA samples tested (29 animals crusts and their cell cultures) were positive in the qPCR. The qPCR was able to detect the DNA of at least 10,000 times dilution corresponding to 0.056 ng of DNA. It is believed that with the additional qPCR validations reported in this study, it can be used for differential diagnosis in the health surveillance of PNEFA.