Background: HIV/AIDS is a serious global health problem with an adverse impact on human health and his socioeconomic status in different countries. Objectives: The aim of the present study was to estimate the prevalence of HIV infection in Mashhad city. Methods: This cross sectional study was performed since May to September 2009 in Mashhad, Iran. A total of 1,678 individuals ranged 1-90 years of age were selected randomly from different geographical regions of the city, proportionate to sex and age distribution of the population according to 2006 census. Enzyme-linked immunosorbent assay (ELISA) was used to screen anti-HIV antibodies and the positive samples were confirmed byWestern Blot (WB) assay. In anti-HIV positive cases, antibodies to human Tlymphotropic virus type 1 (HTLV-1) and hepatitis C virus (HCV) as well as the surface antigen of hepatitis B virus (HBV) were evaluated by the ELISA Kits. Results: Atotal of 1,651serumsamples were analyzed for anti-HIV antibodies. A total number of 751 of participants were males (45.5%) and 900 were females (54.5%). The mean age was 27.9 +/- 19.0 and 30.0 +/- 18.0 years, respectively. Anti-HIV seropositivity was detected in 12 cases (0.73%, 95% CI: 0.38 - 1.27 percent). No samples were further confirmed by WB technique, thus the overall prevalence of HIV infection was 0 (95% CI: 0.00 - 0.22 %). No case with co-infection of HBV, HCV, or HTLV-1 was observed in individuals who showed seroactivity for HIV antibodies. Conclusions: This first population-based survey showed no evidence of HIV infection in the general population of Mashhad. It seems that implemented health policies and strategies have contributed to this low prevalence and this shall be continued.