In this study we explored how picoplankton community structure and diversity varied along three cascade oligo-mesotrophic reservoirs of the Limay River (Patagonia, Argentina): Alicura, Piedra del Águila and Ramos Mexía. We analyzed the spatial changes, covering lotic and lentic stretches along a gradient of 262 km from Andes to steppe, and we also sampled the main affluent of the Limay River (Collon Cura). In all sampling sites the main limnological variables were measured, and the picoplankton abundance (autotrophic and heterotrophic) was analyzed by flow cytometry. The bacterial biodiversity was assessed using high throughput sequencing Illumina MiSeq. We expected an increase in the trophic state along this series of cascade reservoirs, which would determine spatial differences in the structure of the picoplankton communities. We also hypothesized that the lotic and lentic conditions along the system would influence the bacterial composition. The results showed a slight increase in trophic state together with an increase in overall picoplankton abundance downstream, towards Ramos Mexía Reservoir. Picocyanobacteria were represented by phycoerythrin-rich cells all along the system, in accordance to the pattern described for oligotrophic aquatic ecosystems. Multivariate analyses based on bacterial OTU composition and environmental variables showed a spatial ordination of sites following the trend of increasing trophic state downstream. Molecular analyses of bacterial OTU diversity also showed an increase in richness and a decrease in evenness at the lotic stretches, and the opposite pattern in the reservoirs, suggesting that water retention time may play a role in structuring the community composition.