The records of Leptospira spp. in bats from México are scarce. It is known that several species of bats are hosts of pathogenic Leptospira spp.; therefore, they participate in the epidemiological cycle and can generate transmission scenarios to people and animals. The aim was to detect the DNA of Leptospira spp. in bats captured in four sites from the Yucatán Peninsula, México. A kidney fragment was collected from the bats and used to extract genomic DNA. Using a PCR, the DNA of Leptospira spp. was detected. Fifty–four bats of the species Desmodus rotundus, Mimon cozumelae, Pteronotus mesoamericanus, Pteronotus fulvus, Nyctinomops laticaudatus, Peropteryx macrotis, Molossus nigricans, Molossus aztecus, Noctilio leporinus, Saccopteryx bilineata and Mormoops megalophylla were studied. The PCR yielded a total frequency of Leptospira spp. of 44.4% (24/54, 95% CI 36.2–71.7%). Species with at least one positive individual were D. rotundus, M. cozumelae, P. mesoamericanus, N. laticaudatus, P. macrotis, M. nigricans, N. leporinus, M. aztecus, and S. bilineata. A high frequency of Leptospira spp. was found in different species of bats belonging to several trophic guilds. The presence of Leptospira spp. in bats is relevant for Public and Animal Health because the evidence indicates that they can generate zoonotic transmission cycles.