Objectives Fungal culture requires at least 14 days for a final result, compared with 1-3 days for PCR. The study compared a commercial real-time dermatophyte PCR panel with fungal culture in cats in a shelter setting for: (1) diagnosis of Microsporum canis infection; and (2) determination of mycological cure. Methods This was a cross-sectional, observational study of cats with suspicious skin lesions or suspected exposure to dermatophytosis. Hair samples were collected for fungal culture and PCR prior to treatment and at weekly intervals until two negative culture results were obtained. Results One hundred and thirty-two cats were included, of which 28 (21.2%) were culture positive and 104 (78.8%) culture-negative for M canis. PCR correctly identified all culture-positive cats and 92/104 culture negative cats; there were 12 false-positive PCR results. The sensitivity and specificity of PCR were 100% (95% confidence interval [CI] 87.7-100) and 88.5% (95% CI 80.7-93.9), respectively. Data from 17 cats were available for assessment of mycological cure. At the time of the first and second negative fungal cultures, 14/17 (82.4%) and 11/17 (64.7%) tested PCR positive, respectively. Conclusions and relevance PCR showed high sensitivity and specificity for diagnosis of M canis dermatophytosis compared with fungal culture, but was unreliable for identifying mycological cure. False-positive results were relatively common. There were no false-negative PCR results and a negative PCR test was a reliable finding in this study. The ability to rapidly diagnose or rule out dermatophytosis could be a valuable tool to increase life-saving capacity in animal shelters.