A total of 100 blood and 18 urine samples of rodents and suspected dogs were collected from Mumbai, India during 2006-2008. In order to determine the role of animals in transmission of the disease to humans, all the samples were screened retrospectively by real-time polymerase chain reaction for leptospiral DNA and antibodies were detected using microscopic agglutination test. Leptopsiral DNA was detected from two blood and five urine samples from rodents. Of a total of 71 rodent and dog samples investigated for anti-Leptospira antibodies, 14 (19.7%) were positive. Pyrogenes was the predominant serovar found in 100.0% (7/7) and 85.7% (6/7) from suspected canine cases and rodents, respectively; followed by Icterohemorrhagiae, which was found in one rodent sample 14.28% (1/7). The study proves that there is high prevalence of leptospirosis in rodents and dogs in this region, which proves possible role of these animals in transmission of leptospires to humans. Hence it is imperative to necessary control measures to prevent human leptospirosis.