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This study aimed to detect the occurrence of infection by Leishmania spp.in bats from 34 municipalities of Rio Grande do Sul state (RS; southern Brazil) from 2016 to 2021. A total of 109 bats were provided by the Centro Estadual de Vigilância em Saúde of RS, including six species belonged to Molossidae family, six to Vespertilionidae family, and two to Phyllostomidae family. Leishmania spp. was identified using the nested-PCR method by amplifying the SSU rDNA ribosomal subunit gene into four organ pools: (1) the liver, spleen, and lymph node; (2) heart and lungs; (3) skin; and (4) bone marrow of each bat. Three (3/109, 2.7%) animals tested positive for Leishmania spp. The respective PCR-positive organs came from pools 1 and 3. Two bats (Tadarida brasiliensis) were from the municipality of Canoas, and sequences analysis confirms the species identification as Leishmania infantum. In the third bat (Molossus molossus), from Rio Grande, it was not possible to determine the protozoa species, being considered Leishmania spp. Our results indicate that bats can participate in the biological cycle of Leishmania spp. and perform as host, reservoir, and/or source of infection of the protozoa in different areas of RS. More studies will be needed to elucidate the role of these Chiropteras in the circulation of Leishmania spp. This is the first study reporting the occurrence of Leishmania spp. in bats in Rio Grande do Sul state, southern Brazil. [ABSTRACT FROM AUTHOR]