ObjectiveTo evaluate the value of amplification and sequencing of 16S rRNA gene in the identification of clinical rare pathogenic bacteria, and guide the diagnosis and treatment for related clinical infection.Methods12 bacterial isolates that were difficult, or unable to be identified with conventional laboratory methods, or with special phenotypes were collected. The 16S rRNA gene was amplified by polymerase chain reaction (PCR), then  sequenced for identifying bacterial species through BLAST comparison, clinical characteristics of related infection were analyzed. Results12 clinical isolates were all positive for PCR amplification (about 1 500 bp), species were all identified  (similarity  ≥99%), the identified strains were Listeria monocytogenes(n=2), Brucella melitensis(n=2), Fusobacterium mortiferum(n=1), Rothia aeria(n=1), Nocardia farcinica(n=1), Staphylococcus saccharolyticus(n=1), Rhizobium radiobacter(n=1), Prevotella bivia(n=1), Ralstonia mannitolilytica(n=1), and Atopobium vaginae(n=1). The sensitivity of 16S rRNA gene amplification was high, and the minimum detection limit of Escherichia coli ATCC 25922 was 1.5×101 CFU/mL. Clinical data of 12 patients revealed that these strains can cause multisites and multitypes of infection,after patients received targeted antimicrobial therapy, 11 improved, and 1 died.ConclusionSequencing for 16S rRNA gene can rapidly and accurately identify rare, anaerobic, and difficult cultured bacteria, provide laboratory evidence for etiological diagnosis and treatment of different types of infection.