While the clinical manifestations of HSV‐1 and ‐2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV‐1 and ‐2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR‐based assay to accurately distinguish HSV‐1 from HSV‐2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV‐1 and ‐2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV‐1 and ‐2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV‐1 and ‐2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques. J. Med. Virol. 76:350–355, 2005. © 2005 Wiley‐Liss, Inc.