The objective of this study was to identify factors associated with HIV infection among specific population subgroups and complement the HIV surveillance system in Cameroon. Five subgroups (truck drivers, female-sex-workers, university students, health service providers, and residents along Chad-Cameroon petroleum pipeline) were targeted in 2004. Potential participants were approached at their geographically diverse areas and consented to participate in the study. Anonymous blood samples were collected. 4,011 participants were surveyed (35% students, 25% sex-workers, 20% pipeline residents, 12.5% health service providers, 7.5% truck drivers). HIV prevalence was highest among sex-workers [26.4%, (95% CI, 23.6-29.2)], pipeline residents [19.9% (95% CI, 17.1-22.7)] and truck drivers [16.3% (95% CI, 12.3-20.9)] and lowest among health service providers [5.2% (95% CI, 3.4-7.5)] and university students [3.8% (95% CI, 2.9-5.0)]. Risky sexual behaviours were practiced in all subpopulations. Multivariable analysis shows in female-sex-workers that; older age, residing in the grassland region (Northwest and West Provinces) and inconstant condom use were significantly associated with HIV infection. Despite a moderate HIV prevalence in the general Cameroonian population, some subgroups are at much higher risk for HIV transmission and practicing risky sexual behaviours. There is need for expanded prevention and care programs with emphasis on truck drivers, sex-workers and pipeline residents.