The current study was conducted to detect sexually transmitted infectious agents (bacteria and viruses) by developing HRM ( high resolution melting) assay with multiplex real-time PCR using of set primer of each pathogen that was designated by professional software program (Beacon Designer(TM) automates the design of real time primers and probes) for the accurate detection of these infections in Iraqi patients. This study conducted for detection seven different sexually transmitted pathogens (3 bacteria, 3 viruses, and one parasite) among 150 suspected patients as married couples. The results of RT-PCR with HRM assay of the positive controls demonstrated different melting curves, where each melting peak represent the amlification of one of the seven pathogens, also the melting peak of the internal control (housekeeping gene). This experiment included three panels, the panel one represents the optimal melting peak for the positive control of Trichomonas vaginalis, Mycoplasma hominis, Cytomegalovirus the melting temperature each of them was 80.0℃, 84.90℃ and 88.0℃ respectively.