The structure of fish assemblages in lotic systems usually varies from headwaters to the mouth, related to geomorphological and physicochemical changes. We evaluated the main processes that structure the fish assemblages on spatial, seasonal, and decadal (2009-2010; 2019-2020) scales and relationships with environmental variables in two low-impacted Neotropical Pampean streams tributaries to the Río de la Plata estuary (RdlP). Environmental variables related to the longitudinal variation in the fish assemblage were depth, macrophyte cover, and altitude, showing higher values in the headwater streams compared to downstreams. Species richness increased towards lower stream sectors due to the migration of riverine species from the RdlP. Beta diversity partitioning analysis showed that the greatest proportion of variability was explained by the turnover component in both the longitudinal and seasonal analyses. The headwater streams showed a more stable composition, consisting of species of the order Characiformes associated with aquatic macrophytes, while the downstream streams recorded greater variability due to the entry of migrant Siluriformes from the RdlP, highlighting the essential ecological function of Pampean streams for RldP fish assemblages. The decadal comparison showed that the longitudinal structure is persistent in the long term regardless of the study period. Therefore, the consistent pattern of longitudinal variation in fish assemblages and its explanatory variables have applicability in the conservation and biomonitoring of lowland streams, serving as reference for recovery efforts.
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