Leptospirosis, an emerging infectious disease caused by bacteria of the genus Leptospira, is thought to be the most widespread zoonotic disease in the world. A first step in preventing the spread of Leptospira is delineating the animal reservoirs that maintain and disperse the bacteria. Quantitative PCR (qPCR) methods targeting the LipL32 gene were used to analyze kidney samples from 124 House mice (Mus musculus), 94 Black rats (Rattus rattus), 5 Norway rats (R. norvegicus), and 89 small Indian mongooses (Herpestes auropunctatus) from five cattle farms in Puerto Rico. Renal carriage of Leptospira was found in 38% of the sampled individuals, with 59% of the sampled mice, 34% of Black rats, 20% of Norway rats, and 13% of the mongooses. A heterogeneous distribution of prevalence was also found among sites, with the highest prevalence of Leptospira-positive samples at 52% and the lowest at 30%. Comparative sequence analysis of the LipL32 gene from positive samples revealed the presence of two species of Leptospira, L. borgpetersenii and L. interrogans in mice, detected in similar percentages in samples from four farms, while samples from the fifth farm almost exclusively harbored L. interrogans. In rats, both Leptospira species were found, while mongooses only harbored L. interrogans. Numbers tested for both animals, however, were too small (n = 7 each) to relate prevalence of Leptospira species to location. Significant associations of Leptospira prevalence with anthropogenic landscape features were observed at farms in Naguabo and Sabana Grande, where infected individuals were closer to human dwellings, milking barns, and ponds than were uninfected individuals. These results show that rural areas of Puerto Rico are in need of management and longitudinal surveillance of Leptospira in order to prevent continued infection of focal susceptible species (i.e. humans and cattle).