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Background: Leptospirosis is the world's most common zoonotic disease. Mitigation and control rely on pathogen identification and understanding the roles of potential reservoirs in cycling and transmission. Underreporting and misdiagnosis obscure the magnitude of the problem and confound efforts to understand key epidemiological components. Difficulties in culturing hamper the use of serological diagnostics and delay the development of DNA detection methods. As a result, especially in complex ecosystems, we know very little about the importance of different mammalian host species in cycling and transmission to humans. Methodology/principal findings: We sampled dogs from five indigenous Kichwa communities living in the Yasuní National Park in the Ecuadorian Amazon basin. Blood and urine samples from domestic dogs were collected to assess the exposure of these animals to Leptospira and to identify the circulating species. Microscopic Agglutination Tests with a panel of 22 different serovars showed anti-leptospira antibodies in 36 sampled dogs (75%), and 7 serogroups were detected. Two DNA-based detection assays revealed pathogenic Leptospira DNA in 18 of 19 dog urine samples (94.7%). Amplicon sequencing and phylogenetic analysis of 16S rRNA and SecY genes from 15 urine samples revealed genetic diversity within two of three different Leptospira species: noguchii (n = 7), santarosai (n = 7), and interrogans (n = 1). Conclusions/significance: The high prevalence of antibodies and Leptospira DNA provides strong evidence for high rates of past and current infections. Such high prevalence has not been previously reported for dogs. These dogs live in the peridomestic environment in close contact with humans, yet they are free-ranging animals that interact with wildlife. This complex web of interactions may explain the diverse types of pathogenic Leptospira observed in this study. Our results suggest that domestic dogs are likely to play an important role in the cycling and transmission of Leptospira. Future studies in areas with complex ecoepidemiology will enable better parsing of the significance of genotypic, environmental, and host characteristics. Author summary: People around the world interact with a wide range of animals, but one of the closest is the domestic dog. Dogs can be reservoirs of several zoonotic infectious diseases, including leptospirosis. The frequent ecological interactions between people, dogs, and wildlife in indigenous communities living in the Amazon basin might increase the complexity of leptospirosis transmission, in comparison with what has been described for other settings. In the Amazon basin, wild animals and domestic animals may act as reservoirs of the pathogen, excreting the bacteria through their urine. In this work, we analyzed serum and urine samples from dogs living within Kichwa communities from the Yasuní National Park in Ecuador. Serum samples were analyzed with MAT and urine samples with qPCR assays that detect the presence of pathogenic Leptospira. Our results suggest that a high percentage of dogs have been exposed to Leptospira. We identified the presence of ten serovars and three different Leptospira species. These findings provide important insights into the epidemiology of leptospirosis in this ecosystem, showing that dogs are a common reservoir and thus likely to play a critical role in the transmission of the disease. [ABSTRACT FROM AUTHOR]