Leptospirosis is an endemic zoonotic disease in Brazil and is widespread throughout rural populations in the state of Rio Grande do Sul. This study aimed to identify presumptive infecting Leptospira serogroups in human and animal cases and describe their occurrences within the ecoregions of the state by animal species. Data for human and animal leptospirosis cases were gathered from the government’s passive surveillance systems and presumptive infecting serogroups were identified based on a two-fold titer difference in serogroups in the microscopic agglutination test (MAT) panel. A total of 22 different serogroups were reported across both human and animal cases. Serogroup Icterohaemorrhagiae was the most common among humans, while serogroup Sejroe predominated among animal cases, particularly bovines. Each ecoregion had a large distribution of cases, with 51% of the human cases in the Parana–Paraiba ecoregion, and 81% of the animal cases in the Savannah ecoregion. Identifying and mapping the serogroups circulating using the One Health approach is the first step for further understanding the distribution of the disease in the state. This study has the potential to aid in guiding public health and agricultural practices, furthering the need for a human vaccine in high-risk populations to complement control and prevention efforts.