The present paper aims to investigate the herpes simplex virus (HSV) infection in neonates. From November 2010 to June 2011, fresh neonatal cord blood was collected from 51 neonates in a Maternity and Children Hospital and transported immediately to our laboratory. After DNA extraction from the samples, nested-polymerase chain reactions (PCR) were performed by using HSV-1 gD gene- and HSV-2 gG gene-specific primers to detect congenital HSV-1 and HSV-2 infections respectively. The PCR products were sequenced toconfirm the HSV infections.. The results indicated that the infection rate of HSV-1 and HSV-2 were 23.53% and 25.49% respectively. The HSV-1/HSV-2 co-infection rate was 7.84%. This study suggests that early diagnosis and treatment of neonatal HSV infection is critical for the prevention of diseases caused by HSV infection.