Ophidiomycosis, an infectious disease of snakes caused by the fungus Ophidiomyces ophidiicola, is known to cause skin lesions and, in some cases, deeper infections, and even death. It has been documented in captive snakes worldwide and in free-ranging snakes in the United States and Europe. Diagnostic limitations have impeded characterization of the epidemiology of this disease and subsequent efforts to improve clinical care and conservation outcomes. The purpose of this research was to evaluate the efficacy of qPCR of snake sheds as a noninvasive diagnostic tool for the detection of O. ophidiicola, compared to swabs prior to and following the shed. We tested shed pieces from grossly observed skin lesions or mid body sections if disease was not obvious, and matched skin swabs from 68 animals using a qPCR assay specific for O. ophidiicola. There was nearly complete agreement between the qPCR results of sheds and swabs (Cohen's kappa = 0.97) and sheds were 100% sensitive and 97% specific for apparent ophidiomycosis, with a 97% negative predictive value and a 100% positive predictive value. Our results indicate that qPCR of snake sheds is a reliable detection method for the presence of O. ophidiicola, as this technique has high agreement with peri-ecdysis skin swabbing. Although this technique can be used for noninvasive pathogen detection in captive individuals with known histories, results from snake sheds from free-ranging animals with no knowledge of associated clinical signs should be interpreted with caution and additional surveillance is necessary in these cases.