Taiwan is in the subtropical zone and has typhoons every year. Leptospirosis is an endemic disease in Taiwan, and feline leptospirosis in Taiwan remains unknown so far. From January, 2010, to September, 2011, 233 cats in south Taiwan (159 stray cats and 74 household cats) were sampled in this research. Leptospira antibody titer was detected by the serology gold standard, the microscopic agglutination test (MAT). Both serum and urine were examined for Leptospira DNA by polymerase chain reaction (PCR) with two sets of primers. In this study, the serological survey showed 21 (9.3%) examined sera contained antibodies specific for pathogenic Leptospira serogroups. The results of PCR revealed that 25 (19.1%) serum and 80 (67.8%) urine samples were found positive for leptospiral DNA sequences. All products amplified from PCR reactions were sequenced by an automated method for further confirmation. This is the first study concerning the epidemiology of pathogenic Leptospira in stray and household cats' urine, and the results demonstrate that some of the cats are susceptible to pathogenic Leptospira and have the potential to shed pathogenic Leptospira into the environment. This could be an issue of public health.