<b><i>Background/Aims:</i></b> To estimate the clinical value of bacterial detection in peritoneal dialysis-associated peritonitis (PDAP) by multiplex real-time polymerase chain reaction (RT-PCR). This study was undertaken to evaluate multiplex RT-PCR for identifying clinically significant bacteria in PDAP. <b><i>Methods:</i></b> Seventy peritoneal dialysate specimens were collected and traditional bacterial culture and universal primer RT-PCR detection of the bacterial were used. <b><i>Results:</i></b> The positive rate of traditional culture method was 65.71% (46/70) and that of universal primer RT-PCR was 81.42% (57/70). For 6 clinical commonly pathogenic bacteria, multiplex, and monoplex RT-PCR all detected 38 positive ones within the 57 specimens that were detected positive by universal primer RT-PCR. The results of the 2 methods were completely identical. Detecting bacteria by universal primer PCR and Monoplex RT-PCR needs 4–5 and 6–9 h, respectively, while multiplex RT-PCR needs less than 3 h. <b><i>Conclusion:</i></b> Our results demonstrated that the multiplex RT-PCR can detect several kinds of bacteria simultaneously and it is also more practical and convenient than monoplex RT-PCR.