Abstract Background Herpes simplex viruses exist as two major serotypes, type 1 (HSV-1) and type 2 (HSV-2). Determination of type, either HSV-1 or HSV-2, is important in accurate diagnosis and clinical control of transmission. Several tests are available for typing HSV, including a monoclonal antibody specific for glycoprotein G and several PCR assays. Findings A clinical isolate was identified as herpes simplex virus, but tested negative for both HSV-1 and HSV-2 antigens using type-specific monoclonal antibody assays. The isolate was determined to be HSV-1 by PCR analysis. A mutation which likely caused the monoclonal antibody non-reactivity was found in glycoprotein G. Phylogenetic analysis revealed two groups of HSV, one with the mutation and one without. Three population studies examining mutations in HSV-1 glycoprotein G were analyzed by chi-squared test. To this point, the epitope which the monoclonal antibody recognizes was only found in HSV-1 isolates from human European populations (p < 0.0001). Conclusions These findings suggest that the PCR-based methods for HSV typing may be more useful than the standard monoclonal antibody test in areas of the world where the variant in glycoprotein G is more prevalent.