The intensification and expansion of changes in land use and land cover (LULC) can reduce the availability and quality of natural habitats and ecosystem services (ES). These changes have generated environmental damage in different parts of the world, especially in biomes more susceptible to modifications, such as the Pampa biome in the extreme south of Brazil. The Pampa biome has been neglected by environmental protection laws, despite its broad ecosystem and social importance. In this study, we used InVEST models to map and quantify five ES provided (i.e., water supply, carbon stock, groundwater stock, sediment retention and habitat quality) by 14 watersheds distributed in the Brazilian Pampa biome as well as determine ES hotspots by summing the areas with high provision of ES. We identified low availability of water supply and groundwater stock in practically the entire study area. High sediment retention and carbon stock were reported in areas with the presence of native vegetation. In addition, despite the large degraded areas, we observed high habitat quality associated with native vegetation in all studied watersheds. The hotspots varied spatially in the study area according to the proposed scenarios, the supply and the overlap of ES. Scenario 1 (>50% of ES) presented a larger area of high provision of ES. In contrast, scenario 2 (>75% of ES) presented smaller patches of areas with high provision of ES distributed across different watershed. We observed that large territorial extensions with high and medium provision of ES are vulnerable to the negative effects of LULC. Our study presented scenarios that indicate areas of high provision of ES, contributing to a more practical application, being a simplified and useful tool that can assist conservation and sustainable policies.