Herpes simplex virus (HSV) distributing worldwide, belongs to family Herpesviridae, subfamily alphaherpesvirinae. There are 2 serotypes, i.e., HSV-1 and HSV-2. The initial infection starts by using glycoprotein D (gD) contact to host cell surface receptors resulting to viral entry by membrane fusion. Moreover, gD can induce the production of neutralizing antibody. Therefore, mutations of HSV gD are critical for viral growth in cells which might affect cell pathology and pathogenesis of diseases. The objective of this study is to investigate the variations of HSV gD. One hundred samples were selected by using systemic sampling from 256 clinical specimens during the year of 1999 – 2010. Only 70 samples were successfully isolated. They were typed by Real time PCR revealing 37 were HSV-1 and 33 were HSV-2. After that, gD gene was amplified and DNA sequencing. The results were compared to the sequences previously reported in GenBank. From nucleotide sequencing data, HSV-1 gD of 13 patterns were different from HSV-1(KOS) reference strain. While HSV-2 gD showed 9 patterns differed from HSV-2 (HG52) reference strain. Mutation was the major mechanism of variation. Only 6 positions had non-synonymous amino acid changes. After phylogenetic tree was constructed, the variations of HSV-1 gD and HSV-2 gD were distinctively separated. However, intratypic variation was very low. In conclusion, HSV gD from clinical specimens had low variations. HSV-1 gD had more divergent than HSV-2. The major cause of variation was synonymous point mutation. Therefore, HSV gD is a good candidate for vaccine development.