As atmospheric nitrogen (N2) cannot be directly utilized, it must be reduced to ammonia (NH3) by the community of nitrogen-fixing bacteria. The gene encoding nifH is largely unique to nitrogen fixing bacteria, as it converts atmospheric nitrogen into ammonia by producing dinitrogenase reductase enzyme. In the present study, water sample from greenwater system were analyzed using nifH gene as a biomarker. Metagenomic clone library was constructed for nifH genes. Clones exhibit 83-98% similarity at nucleotide level and 88-98% similarity at amino acid level with dominating cluster of gamma proteobacteria, other clusters of beta proteobacteria (Dechloromonas aromatica and Zoogloea oryzae), alpha proteobacteria (Bradyrhizobium japonicum), delta proteobacteria (Desulfovibrio sp.) and uncultured bacteria. Present study sheds light on the high level of species richness in the nitrogen fixing bacterial population in greenwater system of coastal aquaculture.