Wetlands are heterogeneous environments that harbor several species. In spite of playing a key role in aquatic ecosystems, macrophytes are the most neglected group in limnological studies. In the Brazilian subtropical highland grasslands, freshwater wetlands occur intermingled with native grasslands. Our objective was to assess species richness and variation in floristic composition along transects. We sampled 324 units to compare floristic similarity between and within areas based on non-metric multidimensional scaling (NMDS). We recorded plants of 40 families, 88 genera, and 133 species, out of which 17 are endangered. The richest families were Poaceae, Cyperaceae, and Asteraceae. The life forms found included amphibious, emergent, and fixed submerged types. The NMDS pointed to dissimilarity between and within areas. We observed the formation of two zones. In the outer zone, we detected seven indicator species and 17 exclusive species. In the inner zone, we detected five indicator species and 29 exclusive species. Our study suggests that wetlands are species-rich, heterogeneous environments characterized by different zones. The knowledge of biodiversity and presence of indicator species is key to the elaboration of strategies for the restoration and conservation of freshwater wetlands.