Background Endophthalmitis is a serious complication of intraocular surgery.Conventional identification methods for bacteria are becterial culture and smear method,but these laboratory tests spend a long time and have low positive rates.16S rDNA is the bacterial chromosome encoding ribosomal RNA sequences,and it is determined that 16S rDNA sequencing has a high specificity for the identification of bacteria.Objective This study was to identify the infectious bacteria from aqueous humor or vitreous body in the eyes with endophthalmitis by 16S rDNA sequencing technique,and to investigate the diagnosis efficency of 16S rDNA sequencing technique on bacterial endophthalmitis.Methods Anterior chamber fluid (0.1-0.2 ml) or vitreous humor (0.5-1.0 ml) specimens were collected from 5 eyes of 5 patients with endophthalmitis in Qingdao Eye Hospital from June to December 2015 and used for high throughput sequencing,bacterial culture and smear,respectively.Bacteria DNA was extracted from the specimen with D3096-01 trace DNA kit for the amplification of V3-V4 region of 16S rRNA gene and sequencing of hypervariable region of all microbes in the samples by MiSeq Illumina Sequencing Platform.Then the bioinformatic analysis including analysis of taxonomy,abundance and alpha diversity were performed.Nucleasefree water of 50 μl in the centrifuge tube was used as control.Results Five aqueous humor or vitreous body samples were collected,and the positive results were exhibited by smear examination,with the Gram positive bacilli in the trumatic endophthalmitis eye and Gram negative bacilli in the filtering bleb infectious endophthalmitis eye,and all culture results were negative.16S rDNA squencing showed the positive outcomes in all the 5 samples.The high abundent nacteral genuses were Staphylococcus (65.28％),Streptococcus (18.90％) and Pseudomonas (12.76％) in the trumatic endophthalmitis eye;the major components of sample were Pseudomonas (53.68％),Acinetobacter (8.62％) and Limnobacter (5.96％) in the eye with acute endophthalmitis occurring at 2 days following cataract surgery;the major components in the filtering bleb infectious endophthalmitis eye were Moraxella (88.89％) and Pseudomonas (9.52％);the Pseudomonas was major components in the later-onset endophthalmitis eye (84.63％) and the eye with acute endophthalmitis occurring at 1 day after cataract surgery (97.89％).Conclusions A distinct advantage is found in 16S rDNA sequencing technique for the indentification of the pathogenic bacteria in endophthalmitis eyes due to its high positive rate in comparison with bacterial culture and smear method.