Objective: Leptospirosis is a worldwide zoonotic disease and well recognized infectious disease of horses. Equine leptospirosis is to cause the birth of weak foals, neonatal deaths and abortion after pregnancy period. Studies on leptospirosis in horses are generally investigations of urine samples and serological studies. Although leptospirosis causes reproductive disorders, genital sample studies that may be a source of infection have been ignored. The aims of this study were to study the prevalance of Leptospira by PCR using vaginal swab from apparently healthy horses. Methods: A total of 92 vaginal swab samples were collected and transferred to the bacterial diagnosis laboratory of Selcuk University Veterinary Faculty. The vulva and vagina were cleaned before swab samples were taken. All samples were stored in the refrigerator at -20°C and taken to the laboratory for processing. These samples were sent to the laboratory under cold chain conditions. DNA was extracted from suspicious samples and conventional PCR was used to detect Leptospira spp. Specific primers were selected and PCR was finalized for Leptospira spp. Results: As a result of the study, it was found that out of 92 mare’s vaginal swab samples 7 (7.6%) were positive for Leptospira spp. This study is the first report from our country due to the detection of Leptospira spp DNA in asymptomatic mares. Conclusion: It revealed that the Leptospira PCR positive mare were not showing any signs and symptoms. When the results and observations were evaluated, it was thought that Leptospira PCR positive mares could play a role as a carrier in the transmission of leptospirosis. Our study is one of the rare studies on mares carrying the possible causative agent. Detection of leptospirosis by PCR can be considered as a reliable method for early detection of Leptospira shedding in asymptomatic animals. In addition, molecular studies from vaginal swab samples were observed as a rapid and definitive diagnostic option, considering the difficulty of isolation of the Leptospira agent and possible contaminations. According to the results of our study, it is recommended to reevaluate the control measures against the disease and to carry out molecular characterization and vaccination studies in risky areas.