Objective To establish a multiplex polymerase chain reaction （M-PCR） assay for simultaneous detection of pathogens causing genital ulcer disease （GUD）. Methods Based on the gene-specific region of the following pathogens: Chlamydia trachomatis omp1/ompb

herpes simplex virus （HSV） DNA polymerase

Treponema pallidum tpp47

Haemophilus ducreyi 16s rRNA

four sets of primers were designed and an M-PCR assay was developed to detect four pathogens in one test. The assay was evaluated with diagnostic result of golden standard for each pathogen. Results Of the 51 clinical samples

M-PCR showed slightly higher positive rate （47.1%） of HSV than cell culture （23.6%）.Meanwhile

the positive rate of T. pallidum detected by M-PCR and dark-field microscopy was 19.6% （10/51） and 15.7% （8/51）

respectively. Only one sample was positive for H. ducreyi and no sample was positive for C. trachomatis detected by both M-PCR assay and culture. Conclusion This primary study indicated that M-PCR assay can simultaneously and rapidly detect the four etiologic pathogens causing GUD.