Denitrification is a process of reduction of nitrate to dinitrogen by facultative anaerobic microorganisms, which have functional genes encoding denitrification enzymes (reductases). The nosZ gene encoding the enzyme that reduces nitrous oxide to dinitrogen was utilized in this research, as molecular marker for denitrifying communities into low oxygen waters of Prado reservoir. Our objective was to analyze and compare the composition (richness and abundance) of nosZ-type denitrifiers in relationship with physicochemical variables (oxygen, pH, temperature, nitrate, nitrite and ammonium) in three areas of this dam: Isla del Sol, Lozanía and Tomogó which are distant and have different anthropogenic influences. For this, we performed DNA extraction, amplification, 454 pyrosequencing and phylogenetic analysis of nosZ gene. The Chao1 estimator and Shannon index were used for compare richness and diversity of nosZ gene; and the relationship between compositions of operational taxonomic units (OTU) with physicochemical variables was established by canonical correspondence analysis (CCA). In the reservoir 45 nosZ-OTUs to species level (80% similarity) were detected. Lozanía had the highest number of OTUs (25) and greatest diversity (SCHAO1: 35; H: 2.0), compared to the other areas sampling. The phylogenetic analysis showed the presence of many OTUs (28 of 45) with low similarity to Proteobacteria group and high similarity with sequences of environmental clones reported previously. The CCA showed that the nosZ-community composition of Prado dam was related with low pH (6.2), oxygen (0.01mg / L) and nitrate (<0.25 mg / L) recorded in the water column.