There is lack of age-specific seroprevalence surveys and identification of factors associated with herpes simplex virus type-2 seropositivity (HSV-2) in rural populations in Colombia. A random sample of 869 women was interviewed about socio-demographic aspects, sexual and reproductive history. Antibodies to HSV-2 were determined by a specific type immunoenzymatic technique (ELISA). Participants had a mean age of 38±16.1 years, 67% were married, 60% monogamous and 47% reported use of condoms. HSV-2 seroprevalence was 19.1% (95% CI: 16.6-21.9) and it was strongly associated with increasing age (Ptrend<0.001). In the logistic regression analysis, women who reported between two or three lifetime sexual partners (OR=2.4; 95% CI: 1.5-3.7), >31 years of sexual activity with regular or occasional sexual partners (OR=4.3; 95% CI: 1.2-15.7) and not using condoms with regular sexual partners (OR=2.1; 95% CI: 1.4-3.3) were more likely to be HSV-2 seropositive. The overall seroprevalence rate of women of Pueblorrico, Colombia, is lower than that reported in other Latin American countries especially in women>45 years. The difference may be explained by higher prevalence of condom use in this population or lower exposure to herpes infection in male as well as females in the past.