Sporotrichosis is an implantation mycosis caused by pathogenic species of Sporothrix schenckii complex that affects humans and animals, especially cats. Its main forms of zoonotic transmission include scratching, biting and/or contact with the exudate from lesions of sick cats. In Brazil, epidemic involving humans, dogs and cats has occurred since 1998. The definitive diagnosis of sporotrichosis is obtained by the isolation of the fungus in culture; however, the result can take up to four weeks, which may delay the beginning of antifungal treatment in some cases. Cytopathological examination is often used in feline sporotrichosis diagnosis, but accuracy parameters have not been established yet. The aim of this study was to evaluate the accuracy and reliability of cytopathological examination in the diagnosis of feline sporotrichosis. The present study included 244 cats from the metropolitan region of Rio de Janeiro, mostly males in reproductive age with three or more lesions in non-adjacent anatomical places. To evaluate the inter-observer reliability, two different observers performed the microscopic examination of the slides blindly. Test sensitivity was 84.9%. The values of positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and accuracy were 86.0, 24.4, 2.02, 0.26 and 82.8%, respectively. The reliability between the two observers was considered substantial. We conclude that the cytopathological examination is a sensitive, rapid and practical method to be used in feline sporotrichosis diagnosis in outbreaks of this mycosis.