Non-forest ecosystems, as natural grasslands from Southern Brazil, are still neglected in conservation policies. Measuring their biodiversity is one of the main steps to generate management strategies for these habitats. This study aims to (i) describe grassland ant richness and composition in Rio Grande do Sul state, and (ii) compare ant communities sampled on the ground and in grassland vegetation, adding to our knowledge of habitat use patterns and vegetation associated species. Six sites were sampled, three belonging to the Pampa biome and three in highland region from the Atlantic Forest biome. Ant fauna was collected once per year in summer during four years in each site with pitfalls traps and sweeping nets. Overall, 29,812 ant individuals were sampled belonging to eight subfamilies, 30 genera e 106 species. The grasslands of Pampa accumulated 91 species and 45 exclusive species, while highland grasslands summed up 61 species and only 15 exclusive species. Species composition differs between biomes as well as between sampling methods. Ant communities sampled from vegetation represented a clear subset of the fauna sampled with pitfall traps, and indication analysis showed only two species associated with this stratum: Myrmelachista gallicola and Pseudomyrmex nr. flavidulus. This study highlights the importance of Southern Brazilian grasslands and the need for specific conservation strategies for the natural grasslands from each biome.