Purpose: To develop RNA based assay - reverse transcriptase polymerase chain reaction (RT-PCR) to detect viable bacteria in intraocular specimens obtained from patients with infectious endophthalmitis. Materials and methods: Thirty-five intraocular specimens (19 vitreous fluid and 16 aqueous humor) collected from patients with typical infectious endophthalmitis were subjected to conventional and molecular microbiological investigations. Culture negative, eubacterial genome PCR positive intraocular specimens were subjected to denaturing high performance liquid chromatography (dHPLC) for separation of mixed genomes and subsequently identified by PCR based DNA sequencing. In parallel, RT-PCR was performed to detect the presence of viable bacteria in intraocular specimens. Results: Among 35 intraocular specimens, single bacterial genome was detected in 9 (25.7%) and two or more genomes in 26 (74.28%) intraocular specimens. Eubacterial genome was detected by RT-PCR in 29 (82.85%) specimens. PCR based dHPLC followed by PCR based DNA sequencing revealed the presence of 65 bacterial genomes in 35 intraocular specimens. Five novel genera namely Terrabacter species, Facklamia species, Xylella fastidiosa, Duganella species and Synechococcus species were detected. Conclusion: RT-PCR serves as a rapid and reliable tool to detect viable bacteria causing endophthalmitis.