Objectives The aims of this descriptive study were to identify risk factors for feline Microsporum canis infection at shelter intake, to describe screening test accuracy, and to refine confirmatory testing time frames. Methods Database records for the general feline population and intake data, medical records and fungal culture logs for cats diagnosed with M canis at a limited admissions shelter were accessed retrospectively for a period of 2 years. Results The feline population at the study shelter had a prevalence of M canis of 1.8% (95% confidence interval [CI] 1.6–2.0%). Kittens were eight (95% CI 4.8–13.5) times more likely to present with dermatophytosis than adults. Although more cats presented with M canis during summer and autumn, season was not significant when the model was controlled for age. Owner-surrendered cats were half as likely (95% CI 0.41–0.77) as transported cats to be diagnosed with M canis. Wood’s lamp examinations had a sensitivity of 66.8% (95% CI 60.2–73.4) and a specificity of 74.8% (95% CI 64.2–85.1) compared with dermatophyte test medium (DTM) culture. In 78.8% (95% CI 61–91) of littermate or household groups with mixed Wood’s lamp results, all cats were DTM culture positive. Under consistent incubation conditions, 202/202 diagnostic DTM plates for M canis-infected cats showed recognizable colony growth before 7 days (median 4 days), and 19/19 fomite carrier cat cultures showed growth before 12 days (median 5 days). Conclusions and relevance Applying the results of this study to shelter protocols could optimize diagnostic approaches and shorten the length of stay for shelter cats and kittens, resulting in streamlined shelter operations and improved feline welfare.