Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that causes a variety of diseases, including an increased risk of developing more severe disease in HIV-infected individuals. In Brazil, there is no information about the molecular epidemiology of HSV-1 infection, especially in HIV-infected individuals. The aim of this study was to perform the genotypic characterization of HSV-1 among HIV-infected patients. A total of 214 serum samples from HIV-positive patients without HSV infection symptoms were enrolled in one of two reference hospitals for HIV infection managing in Rio de Janeiro. The gG and gI genes were analyzed by restriction fragment length polymorphism (RFLP) and full nucleotide sequencing of the US8 (1601 bp), UL44 (1996 bp), and UL23 (1244 bp) regions was performed. A total of 38.3% (82/214) and 32.7% (70/214) of the serum samples tested positive for gG and gI genes, respectively. RFLP analysis classified the HSV-1 as belonging to genotype A. Phylogenetic analysis of the Brazilian samples for the US8, UL44, and UL23 regions demonstrated that the nucleotide identity between Brazilian samples was higher than 97% for all genes. No acyclovir mutation was detected in the patients. The shedding of HSV in the serum samples from HIV-positive patients who were asymptomatic for HSV infection was detected in this work. This is the first report of molecular characterization of HSV-1 in Brazilian samples since there is no previous data available in the literature concerning the genotypic classification and stable distribution of Brazilian strains of HSV-1 in Rio de Janeiro, Brazil.