Interest in the study of infectious diseases of wildlife has grown in recent decades and now focuses on understanding host-parasite dynamics and factors involved in disease occurrence. The black-tailed prairie dog (Cynomys ludovicianus) is a useful species for this type of investigation because it lives in heterogeneous landscapes where human activities take place, and its populations are structured as a metapopulation. Our goal was to determine if colony area, density, and proximity to human settlements are associated with prevalence of antibodies to Leptospira interrogans in black-tailed prairie dogs of northwestern Chihuahua State, Mexico. We captured 266 prairie dogs in 11 colonies in 2009 and analyzed 248 serum samples with the microscopic agglutination test (MAT) for antibody to any of the 12 pathogenic serovars of L. interrogans. Serologically positive test results for only serovars Bratislava, Canicola, Celledoni, and Tarassovi were considered for statistical analysis. Almost 80% of sera were positive for at least one pathogenic serovar (MAT titer ≥1∶80). The highest recorded antibody prevalences were to serovars Bratislava and Canicola. Correlation analysis showed a negative relationship between L. interrogans antibody prevalence and colony area (r = −0.125, P<0.005), suggesting that animals living in larger colonies were at a lower risk than those in smaller colonies. The correlation between the serovar Canicola and distance was negative (r = −0.171, P<0.007), and this relationship may be explained by the presence of domestic dogs associated with human dwellings. This is the first study of Leptospira spp. antibody prevalence in prairie dogs, and it provides valuable insights into the dynamics of leptospirosis in threatened wildlife species. Further studies are needed to evaluate the impact of Leptospira serovars in metapopulations of prairie dogs and other domestic and wild mammals in grassland communities.