Drying River Networks (DRN) are common in the Neotropics, but the relationship between flow cessation, environmental variables, and biological responses is still poorly understood. Under pressing climate changes, streamflow is expected to vary drastically thus affecting diversity and ecological processes driven by hydro-climatic conditions. Aquatic invertebrates, as sentinels of flow fluctuation, are expected to show strong responses to flow cessation in intermittent streams. Here, we use a meta-community approach, to explore the influence of local and regional factors in structuring the composition and spatial variability of freshwater invertebrate communities in three Neotropical biomes: 1) The Chocó Ecoregion in the Pacific lowlands of northwestern Ecuador; 2) The Central High Andean Ecoregion in Bolivia; and 3) The Caatinga Ecoregion in Brazil. In each DRN, we sampled 20 points (including pool and riffle habitats) in six occasions during 2021, covering the wet, transitional, and dry periods. We identified invertebrates to family level and compared the communities from each biome. Our preliminary results indicate that local environmental variables were markedly different between seasons and biomes. In contrast, local invertebrate community composition (alpha diversity) varied significantly between biomes but not between seasons. At the landscape scale, invertebrate density, biomass, and community composition changed between seasons with different eco-hydrological conditions. Due to their adaptations and habitat preferences, some species were indicative of sites exhibiting flow intermittency, whereas other were indicative of downstream perennial stream reaches. Metacommunity analysis showed that, in both seasons and in all biomes, community dissimilarity was significantly correlated with environmental distance, but not with distance. Community dissimilarity exhibited a positive correlation with distance only for families with low dispersal ability. Our findings suggest that environmental filtering acts as the main driver of change in these intermittent river networks in all three Neotropical biomes.