Leptospirosis is a neglected and severe zoonotic bacterial disease that affects canine populations worldwide, and it is present in animals from different settings, such as urban and rural environments. Therefore, a cross-sectional study was conducted on owned domestic dogs from urban and rural origins in southern Chile. The study aimed: 1) to estimate the true prevalence of pathogenic Leptospira in dogs from urban and rural environments in southern Chile, 2) to determine the serovars circulating in an endemic area of Chile, 3) to assess potential risk factors associated with seropositivity in dogs from urban and rural environments. Blood samples from 706 canines were collected, and the serum was tested with Micro-Agglutination-Test (MAT), using a panel of 13 serovars. A Bayesian approach was applied to estimate True prevalence (TP). In addition, dog owners answered a questionnaire that had prompts regarding dog characteristics and potential risk factors. The effect of these factors on the risk of being infected was analysed using a logistic regression model. The TP for urban dogs was 9.4% and 9.1% for rural animals. However, seroprevalence was higher in dogs with different individual, lifestyle, and habitat characteristics regarding their origin. For example, in urban animals, those living in places with rodents roaming near houses and dogs with rodent hunting habits had decreased risk for the seropositivity in MAT. On the other hand, in rural dogs, those of a recognized breed, with contact with cattle and sheep, living with more than one cat at home and living in places close to natural water sources have more risk to seropositivity in MAT. The presence of anti-Leptospira antibodies is widespread in pet dogs in urban and rural environments in southern Chile, and individual, lifestyle and habitat characteristics of the animals, according to the origin, are associated with the seropositivity in MAT. Therefore, the adoption of preventive measures is urgent, in addition to increase the awareness either in public health institutions, practitioners and companion animals’ owners, considering the zoonotic potential of Leptospira infection and the close contact between people and their pets.