<p>The Iberá Wetlands represent one of the largest and least disturbed freshwater ecosystems in South America. Although there has been a long history of research in these wetlands, their ostracod fauna remains unexplored. This study provides the first characterization of ostracod assemblages, analyzing their density, richness, and composition in relation to environmental drivers. Standardized sampling was conducted across littoral, limnetic, and benthic zones using hand nets and dredges. Assemblages were compared between wetland hydrological regimes (lentic versus lotic environments) and two dominant macrophyte species with contrasting architectures (<i>Egeria najas</i> and <i>Pontederia azurea</i>). Although no significant differences in richness were found between lentic and lotic environments, some species exhibited clear habitat preferences. Macrophyte architecture also influenced assemblage composition, with distinct species associated with each plant type. Our findings establish the first regional baseline and constitute valuable data for future monitoring and comparative studies, particularly in the context of global change and the increasing anthropogenic pressures on subtropical freshwater ecosystems.</p>