<p>This study aimed to elucidate the relationships between phytobenthos and natural environmental gradients in the least disturbed stream reaches of the Pampean plain. We hypothesized that if basal conditions are maintained, variables associated with the latitudinal gradient, such as irradiance and temperature, should be the main drivers of phytobenthos and that functional trait-based approaches would be more suitable than taxonomic ones for describing these relationships. Eighteen stream reaches belonging to different hydrological basins were selected, based on expected morphological and hydrological characteristics for undisturbed plain streams, with lowest impact from land uses in the surroundings. Traditional taxonomic approach and two functional perspectives based on species traits were used to analyse the responses of phytobenthos to environmental variables of local and regional variability. Due to the favourable conditions given by nutrient availability and low current velocity, we consider and verify through several constrained multivariate analyses, that the natural gradient of temperature and irradiance is one of the main structuring factors of the phytobenthos community. Both taxonomic and functional approaches provided explanations of different aspects of the ecosystem. These results provide a reference baseline to restoration and management policies at a global level, particularly in temperate grasslands ecosystems facing significant anthropogenic pressures.</p>