Biogas digesters contain microbial assemblages that process a mass of extracellular polymeric substances from animal manure and domestic wastewater; however, due to the limitation of available technology in cultivation of majority of the micro-organisms in biogas digesters, the enzymatic potential of these microbial communities remains largely unexplored. In this study, to evaluate subtilase gene diversity in a biogas digester, the partial sequences of the gene were directly amplified from the metagenomic DNA by using consensus-degenerate primers. The desired PCR products were cloned into pGEM-T Easy vector, and thirty positive clones were chose for Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, from which thirteen distinguished patterns were obtained and then sequenced. Phylogenetic analysis showed that ten out of the thirteen sequences were related to the subtilase genes in GenBank and were grouped into three families of the subtilases superfamily. The nucleotide sequences analysis through BLAST search revealed that none of the partial genes the authors isolated showed significant similarity against the non-redundant Nucleotide database of NCBI. Meanwhile, the deduced amino acid sequences of ten partial subtilase genes showed moderate identities to the previously identified sequences in GenBank, with a range from 39 to 61%. Collectively, the data indicate that there is a great diversity of subtilase genes in the biogas digester; and may be a rich reservoir for novel subtilase genes.