The population of men who have sex with men (MSM) has been largely ignored in HIV-related policies and programming in Mozambique and there is little information about the contribution of MSM to the HIV epidemic. An integrated biological and behavioral study among MSM using respondent-driven sampling was conducted in 2011 in Maputo, Beira and Nampula/Nacala. Men who reported engaging in oral or anal sex with other men in the last 12 months answered a questionnaire and provided a blood sample for HIV testing. The prevalence of HIV was 8.2 % (Maputo, n = 496), 9.1 % (Beira, n = 584) and 3.1 % (Nampula/Nacala, n = 353). Prevalence was higher among MSM ≥ 25 vs. 18-24 years: 33.8 % vs. 2.4 % (p < 0.001), 32.1 vs. 2.8 % (p < 0.001), and 10.3 vs. 2.7 % (p < 0.06), in each city respectively. The difference in prevalence demonstrates the need to increase prevention for younger MSM at risk for HIV and ensure care and treatment for older HIV-infected MSM.