Abstract: In the present study, a primer pairs in the HIV-1 POL gene were evaluated by performing RT-nested PCR and PCR and results were compared with universal GAG primers. In this study 80 HIV-1 seropositive patients from the patient cohort of the infectious disease division at Imam Khomeini Hospital in Tehran and 40 HIV-1 seronegative blood donors were evaluated by performing PCR and RT-PCR with the widely used SK primers (gag) and new designed primer set. Determination of PCR and RT-PCR sensitivity (copy number) was performed. The newly designed POL primer pairs was shown to be highly sensitive (100%) and specific (100%) for detection of HIV-1 RNA in Iranian patients. New primer designed detected HIV-1 RNA and DNA in all 80 plasma samples and 69 PBMC samples. SK38/39 could only detect HIV-1 RNA and DVA in 54 plasma samples and 37 PBMC samples, respectively. The optimum PCR profile for a specific primer pairs was defined as that which detected one copy of proviral plasmid DNA and 500 copies of viral RNA. Present results demonstrated that the use of primers designed for highly conserved regions of viral genome along with appropriate optimization of the test, leads to results with higher sensitivity. The high sensitivity of the developed RT-PCR allows its use as a qualitative screening test in patients receiving antiretroviral therapy in Iran.