Abstract: Background and Objective: The number of Leptospirosis outbreak in horses was increasing in recent years in Chennai, India. There are reports in other parts of the world about the prevalence of this organism in clinically healthy horses. Hence, the present study was undertaken to assess the seroprevalence of Leptospira spp. in clinically healthy horses and to find out the association among age, gender and serovar type in Chennai, India. Methodology: Sera samples collected from horses (n =162) in and around Chennai were tested for a battery (n = 12) of Leptospira antigens using Microscopic Agglutination Test (MAT). Results: Out of 162 (Male: 40, Female: 122) samples tested, 126 (77.77%) were positive out of which 19.84% were males and 80.16% were female horses, respectively. Among the 40 male horses 25 (62.50%) were positive and among the 122 females 101 (82.79%) horses were positive. Conclusion: The most of the healthy horses in Chennai, India are seropositive carriers for asymptomatic Leptospiral infection. It should be viewed seriously under zoonoses perspective.