The aim of this study was to investigate the presence of Herpes simplex virus (HSV) type 1 and type 2 DNA from the clinical samples sent to our routine laboratory, by real-time polymerase chain reaction (PCR). A total of 328 samples collected from 306 female and 7 male patients who were admitted to different outpatient clinics were included in the study. The samples included 235 cervical swab samples (of which 150 were from pregnant women), 77 amniotic fluid, 8 blood, 6 cerebrospinal fluid (CSF), one pericardial fluid and one cervical biopsy. DNA extraction were performed with High Pure Viral Nucleic Acid Kit (Roche, Germany) and amplified in Light Cycler (Roche, Germany) with a commercial amplification mix (Metis Biotechnology, Ankara). HSV-DNA positivity were found in 2.1% of the cervical samples (three of 150 pregnant and two of 85 non-pregnant women), two of the blood samples and one of the CSF sample, while there were no positive result for the other clinical specimens. It can be concluded that, real-time PCR would be preferred in conditions requiring rapid diagnosis such as HSV infections of central nervous system and HSV suspected infections of immunosupressed patients, as a rapid and practical method.