BACKGROUND: We report on HIV acquisition and its associated risk factors in 5 fishing communities on the shores of Lake Victoria in Uganda. A cohort of 1000 HIV-uninfected at-risk volunteers aged 13 to 49 years were recruited in 2009 and followed up for 18 months. METHODS: At enrollment and semiannual visits, socio-demographic and risk behavior data were collected through a structured questionnaire and blood samples tested for HIV and syphilis. Detailed life histories were collected from 78 volunteers using in-depth interviews. RESULTS: Of the 1000 volunteers enrolled, 919 (91.9%) were followed up, with 762 (76.2%) reaching the study end points (either seroconverted or completed 4 visits). There were 59 incident cases in 1205.6 person-years at risk (PYAR), resulting in an incidence rate of 4.9 (95% CI = 3.8 to 6.3) per 100 PYAR. The highest HIV incidence rates were among those working in bars (9.8/100 PYAR [4.7-20.6]), protestants (8.6/100 PYAR [5.8-12.7]), those aged 13 to 24 years (7.5/100 PYAR [5.2-11.0]), and new immigrants (6.6/100 PYAR [4.9-8.9]). HIV infection was independently associated with being young (adjusted hazard ratio (aHR) = 2.5 [95% CI = 1.3-4.9]), reporting genital sores/discharge recently (aHR = 2.8 [1.6-5.0]), regular alcohol consumption (aHR = 3.3 [1.6-6.1]), use of marijuana (aHR = 2.9 [1.0-8.0]), cigarette smoking (aHR = 3.6 [1.4-9.3]), and religion (compared with Catholics, Protestants had aHR = 2.7 [1.4-5.3] and Muslims had aHR = 2.3 [1.1-4.8]). CONCLUSIONS: These fishing communities experienced high HIV infection, which was mainly explained by high-risk behavior. There is an urgent need to target HIV prevention and research efforts to this vulnerable and neglected group.