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This study represents the first analysis of the bacterioplankton community structure from a freshwater reservoir of Argentina using amplification of the entire 16S rDNA gene. It includes the description and the phylogenetic relationships of the bacterioplankton community from the photic and aphotic layers of the Río III Reservoir in Córdoba, Argentina. The classical ecological approach indicated that the photic layer had greater diversity whereas the aphotic layer had a better distribution of species and higher abundance. Nevertheless, when the microbial communities in both layers were compared using phylogenetic information, this analysis indicated that both environments were similar and that neither was enriched for any particular lineage. The phyla present in the Río III reservoir were Acidobacteria, Actinobacteria, and Proteobacteria and the 2 dominant species in both layers were "Candidatus Planktophila sp." (class Actinobacteria) and Polynucleobacter sp. (class Betaproteobacteria).