<p>Wetlands are dynamic ecosystems shaped by hydrological and climatic variations, which directly influence species distribution and community structure. Aquatic macrophytes play a key ecological role in these environments, yet their distribution patterns remain understudied, especially in the subtropical wetlands of southern Brazil. Among them, the <i>Alismataceae</i> family stands out for its notable diversity and wide geographic range. This study aimed to compare species richness and composition across geographical units (such as biomes and hydrographic basins) in the subtropical wetlands of southern Brazil, as well as to identify indicator species and life forms associated with each region. We compiled occurrence records from online databases, herbarium specimens, and field surveys, classifying the species by biome, hydrographic basin, life form, and habitat. Species richness and composition varied significantly between biomes, with the Pampa biome exhibiting higher richness than the Atlantic Forest. Species with broad distribution across the region showed low habitat specificity. Indicator species analysis revealed that <i>Sagittaria montevidensis</i> and <i>Hydrocleys nymphoides</i> are characteristic of the Pampa biome, although both species also occur in the Atlantic Forest of Rio Grande do Sul. Most species exhibited more than one life form, with amphibious and emergent types being the most common, traits likely influenced by water depth and adaptive plasticity. Our findings enhance the understanding of <i>Alismataceae</i> distribution patterns and ecological strategies in subtropical wetlands, supporting future conservation and management initiatives in subtropical wetlands.</p>