Abstract: OBJECTIVE To investigate the difference between the ability in microbial identification when employing different length of 16S rRNA fragments. METHODS The 16S rRNA sequences of pharmacopoeia control bacteria and related bacteria were obtained from the NCBI, sequences were split into 500 bp, 1 000 bp, or full-length(~1 500 bp) fragments using different primer pairs, and the sequences were compared and annotated by BLAST to obtain similarity for further statistical analysis. RESULTS The 500 bp, 1 000 bp and full length(~1 500 bp) fragments were able to identify all the sequences to genus level, and the identification to species level was enhanced with the increase in fragments’ length, 90.73%, 94.04% and 96.36% sequences were able to be identified by 500 bp, 1 000 bp and full-length fragments(~1 500 bp), respectively. Clostridium spp., Salmonella spp., Escherichia coli and Pseudomonas aeruginosa could be identified with all three fragment lengths which requested by pharmacopeia, while Staphylococcus aureus was best identified using the full-length(~1 500 bp). CONCLUSION The longer length of the 16S rRNA fragment performed better in microorganism identification, whereas the 1 000 bp is recommended in practice due to its advantage in cost. Escherichia coli and Staphylococcus aureus might not be well identified in routine use.