The purpose of this cross-sectional study was to assess the 2009 prevalence of chlamydial and gonococcal infection in 2 populations in a midwestern city in the United States: patients at a public sexually transmitted disease (STD) clinic, and individuals seeking human immunodeficiency virus (HIV) counseling and testing services at an AIDS community organization. We characterized STD prevalence in a random sample of 592 STD clinic patients and a convenience sample of 471 individuals agreeing to STD testing through outreach efforts at the community organization. The STD clinic population was 59% male, 60% black, with 3.1 mean sex partners in the last year. The community organization population was 72% male, 19% black, with a mean of 4.3 partners in the last year. The prevalence of both chlamydial and gonococcal infections was consistently higher in STD clinic patients than at the community organization (18% vs 4%). Prevalence of chlamydial infection was higher than prevalence of gonococcal infection in both populations (chlamydial infection, 3% and 13% at the STD clinic and community organization, respectively; vs gonococcal infection, 1% and 7%, respectively). Factors significantly associated with increased odds of gonococcal/chlamydial infection at the STD clinic include unmarried status, younger age, at least 6 partners in the last year, and unprotected sex in the last year. At the community organization, the only factor significantly associated with increased odds of gonococcal/chlamydial infection was lower educational attainment. Our findings confirm that STD prevalence differs widely by population group. Given these differences, local approaches to STD control should also be carefully targeted to specific subgroups.