Herpes simplex virus type-2 (HSV-2) is the major cause of genital ulcer diseases (GUD) consequently a significant factor for the acquisition and transmission of human immunodeficiency virus (HIV). Despite its importance there is paucity of data regarding the magnitude of HSV-2 in non-HIV infected population in middle-and-low income countries. This study was designed to determine the seroprevalence of specific HSV-2 antibodies among non-HIV population in Mwanza City, Tanzania. A total of 174 non-HIV infected individuals were enrolled and tested for HSV-2 IgM and IgG antibodies using one step HSV-2 rapid Test. The median age of study participants was 28 years (IQR= 23-36 years). Of the 174 enrolled individuals, 22 (12.6%, 95% CI: 7.6-17.5) were HSV-2 IgG seropositive while none of the participants was HSV-2 IgM seropositive. This study shows that a significant proportion of non-HIV infected population is seropositive for HSV-2 antibodies. This might increase the risk of becoming infected with HIV and other sexually transmitted infections.