Aim: The clinical diagnosis of sepsis is difficult, particularly in neonates. It is necessary to develop a rapid and reliable method for detecting bacteria in blood and cerebrospinal fluid (CSF) Polymerase chain reaction (PCR) and reverse hybridization of the 16S rRNA gene would permit fast and sensitive determination of the presence of bacteria and differentiate gram‐positive bacteria from gram‐negative ones in clinical specimens. Methods: We developed a pair of primers according to the gene encoding 16SrRNA found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with PCR, and with reverse hybridization using a universal bacterial probe, a gram‐positive probe and a gram‐negative probe. Results: A 371 bp DNA fragment was amplified from 20 different bacterial species. No signal was observed when human DNA and viruses were used as templates. The sensitivity could be improved to 10T‐12 g. All 26 culture‐positive clinical samples (22 blood samples and 4 CSF samples) were positive with PCR. The gram‐negative and gram‐positive probes hybridized to clinical samples and to known bacterial controls, as predicted by Gram's stain characteristics. Conclusions: Our results suggest that the method of PCR and reverse hybridization is rapid, sensitive and specific in detecting bacterial infections. This finding may be significant in the clinical diagnosis of sepsis in neonates.