Development of a globally effective HIV-1 vaccine will need to encompass Nigeria, one of the hardest hit areas, with an estimated 3.2 million people living with HIV. This cross-sectional Institutional Review Board (IRB) approved study was conducted in 2009-12 at four market sites and two highway settlements sites in Nigeria to identify and characterize populations at high risk for HIV; engage support of local stakeholders; and assess the level of interest in future vaccine studies. Demographic, HIV risk data were collected by structured interviewer-administered questionnaires. Blood samples were tested on site by HIV rapid diagnostic tests, followed by rigorous confirmatory testing, subtype evaluation and testing for HBV and HCV markers in a clinical reference laboratory. Of 3229 study participants, 326 were HIV infected as confirmed by Western Blot or RNA, with a HIV prevalence of 15.4%-23.9% at highway settlements and 3.1%-9.1% at market sites. There was no observable correlation of prevalence of HIV-1 (10.1%) with HBV (10.9%) or HCV (2.9%). Major HIV-1 subtypes included CRF02_AG (37.5%); G (27.5%); G/CRF02_AG (25.9%); and non-typeable (8.9%), with 0.3% HIV-2. Univariate analysis found age, gender, marital status, level of education, and sex under substance influence as significant risk factors for HIV (p<0.001). Educating and winning the trust of local community leadership ensured high level of participation (53.3-77.9%) and willingness to participate in future studies (95%). The high HIV prevalence and high risk of HIV infection at highway settlement and mammy markets make them well suited for targeting future vaccine trials in Nigeria.