Bats are known as potential carriers of different pathogens; these animals have been identified worldwide as an important reservoir of different species of Leptospira. Therefore, there has been an increasing interest in studying leptospirosis in neotropical bats in the Amazon. This region is a fertile ground for zoonotic diseases, given the intense process of deforestation, urbanization, opening of new agricultural frontiers, predatory hunting, effects of climate change, and loss of biodiversity. Based on this, the aim of the present study was to investigate the frequency of infection associated with the genetic characterization of pathogenic Leptospira spp. in bats inhabiting diverse landscapes in the southwestern region of the Amazon. To conduct the study, mist nets were installed to capture bats. Kidney samples were submitted to LipL32-polymerase chain reaction and secY gene sequencing. Our results showed that 21% of neotropical evaluated bats in Southwestern Amazon were infected with Leptospira spp. Positive animals were found in rural, urban, periurban, and control areas. Sanguinivores had the highest frequency of positives, followed by insectivores and frugivorous bats. The species of L. interrogans and a potential new Leptospira species were identified. The frequency of Leptospira in bats was not influenced by landscape type, suggesting these pathogens may not be affected by landscape changes. The findings suggest that bats may serve as potential reservoirs of Leptospira in diverse landscapes. The presence of Leptospira in bats appears to be independent of the type of land use, implying that these pathogens may not be affected by small-scale changes in the environment.