In the United States and Europe, human onchocerciasis is a rare disease caused by zoonotic or anthropophilic parasites in the genus Onchocerca. The zoonotic species identified in focal areas of Europe and United States is Onchocerca lupi, and Onchocerca volvulus, the anthroponotic species, may be found among people who had lived in endemic areas of Africa, the Arabian Peninsula, or Latin America. Onchocerciasis due to O. lupi is an emergent parasitic disease, with limited diagnostic methods, in addition to the lack of information on its biology, transmission, and epidemiology. Cutaneous nodules are the disease's most prevalent manifestation but lack diagnostic specificity. To address the diagnosis of onchocerciasis at reference laboratories, we developed a duplex TaqMan real-time PCR (qPCR) method, targeting the cytochrome oxidase subunit I locus which has species-specific probes to identify and differentiate O. lupi from O. volvulus. We determined the performance of the duplex with a panel of 45 samples: 11 positives for O. lupi, six for O. volvulus, five samples with negative results for Onchocerca spp., and 23 non-Onchocerca nematodes. The duplex qPCR correctly detected 10 of 11 O. lupi- and six of six O. volvulus-positive specimens. The new duplex assay allowed the simultaneous detection and discrimination of O. lupi and O. volvulus in clinical specimens, expediting and facilitating the clinical diagnosis of O. lupi in non-endemic settings where the disease is an infrequent finding.