Objective: Viral Infections are major problems for transplant recipients. Reactivation of viruses is associated with the use of strong immunosuppressive drugs. Herpes simplex virus infections in renal transplant patients however, may become sever without treatment. The aim of this study was to investigate the incidence of HSV-1 and HSV-2 DNA in urine, plasma and peripheral blood mononuclear cells (PBMCs) of renal transplant recipients. Materials and Methods: In a prospective single center study, we followed 58 renal transplant recipients who received a living or cadaveric kidney transplant at Namazi hospital, Shiraz University of medical sciences. Urine and blood samples were collected from each recipient one week before transplantation up to two weeks post-transplantation. Patients were then sampled every four weeks up to 44 weeks after transplantation. Samples were also collected from 37 organ donors and 30 healthy individuals as the control groups of the study. PBMCs were isolated using Ficoll-Hypaque protocol and stored at -20°C until required. Specific DNA extraction methods were employed for every sample type for their ability to remove polymerase chain reaction (PCR) inhibitors from different samples. For PCR amplification, oligonucleotide primers targeting thymidine kinase (TK) gene regions of both HSV-1 and HSV-2 were selected. Discrimination between HSV-1 and HSV-2 amplicons were performed using restriction enzymes AvaI and AvaII. Patients were also examined for any signs or symptoms of renal dysfunction or other related disease. Results: HSV-1 DNA was detected in mononuclear leukocytes of 46.6% of transplant recipients and in 16.2% and 6.6% of living donors and healthy individuals respectively. HSV-1 DNA was only determined in urine samples of 19% of transplant recipients. HSV-2 DNA however, was not detected in any group of subjects including renal transplant recipients. Conclusion: Detection of HSV-1 DNA in mononuclear leukocytes of renal transplant patients and normal population suggests that these cells could be another site for virus latency. HSV-1 viruria on the other hand, is a sign of viral reactivation, which might progress to generalized HSV infection. Although the impact of HSV related disease in renal transplant patients is not well understood and prospective studies are needed to elucidate this further but HSV-DNA PCR in urine or blood samples may be helpful in monitoring and detection of possible related infections and thus, initiate an efficient therapy.