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Worldwide, Leptospira infection poses an increasing public health problem. In 2008, leptospirosis was recognised as a re-emerging zoonosis of global importance with South-East Asia being one of the most significant centres of the disease. Rodents are thought to be the most important host for a variety of Leptospira serovars. Because Bangladesh offers a suitable humid climate for the survival of these pathogenic bacteria, the presence of rodents could be a serious risk for human infection, especially in peri-urban areas or locations where food is stored. In order to gain more understanding of the multi-host epidemiology, a prevalence study was conducted in Comilla, Bangladesh to determine the presence of pathogenic Leptospira species in rodents. Real-time Polymerase Chain Reaction (qPCR) and sequencing showed that 13.1% (61/465) of the trapped rodents were infected with pathogenic Leptospira. Sequencing of the qPCR products identified the presence of three species: Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri. Rodents of the genus, Bandicota, were significantly more likely to be positive than those of the genus, Rattus and Mus. Our results confirm the importance of rodents as hosts of pathogenic Leptospira and indicate that human exposure to pathogenic Leptospira may be considerable, also in places where food (rice) is stored for longer times. This study emphasizes the need to improve rodent management at such locations and to further quantify the public health impacts of this neglected emerging zoonosis in Bangladesh.