Leptospirosis is a widespread zoonotic disease with well-established impacts on human health in tropical and subtropical regions. Although Leptospira spp. are known to readily infect many wildlife species, the understanding of interspecies and climatic variability in patterns of infection in Neotropical mammals is limited. To improve the understanding of this interplay, 85 mammals representing 17 species were sampled from four Colombian zoos along a climatic gradient. Prevalence of the 21 primary serovars against Leptospira interrogans was determined using the microagglutination test. Individuals were considered positive for a given serovar if antibodies were observable at a 1:100 dilution or greater. Overall prevalence was 9.52%, with positive titers to serovar hurstbridge in Carnivora (Canidae); serovar sarmin in Primata (Atelidae); and serovars australis, mini, autumnalis, pomona, icterohaemorrhagiae, and seramanga in Primata (Cebidae). Prevalence was positively correlated with humidity and temperature, with significantly higher prevalence at the site characterized by high humidity, severe flooding because of rainfall, and warm weather throughout the year. All positive animals were classified as clinically asymptomatic, meaning that antibodies from a current or past infection were detected but no overt symptoms were apparent. The diversity of serovars observed and the taxon-specific nature of these associations suggest that the epidemiology of Leptospira transmission is likely to be complex and multidimensional. The strong association observed between prevalence and climate suggests that the important role of climate as an indicator of Leptospira infection risk in humans may also be applicable to wildlife. Future studies in both wild and captive populations of Neotropical wildlife will further elucidate this disease interplay.