Hepatitis B virus (HBV) is a global public health problem and requires specific prevention actions, particularly focusing on the key populations, such as men who have sex with men (MSM). We aimed at assessing the prevalence of HBV infection, among MSM, in a multicity study in Brazil. In 2016, we conducted a survey using a respondent-driven sampling methodology in 12 Brazilian cities. Rapid tests (RT) were performed on 3178 samples from those MSM. Positive results were tested for HBV DNA and sequenced. If negative for HBV DNA, samples were tested for serological markers. The prevalence rate of HBV exposure and clearance was 10.1% (95% CI: 8.1-12.6), and 1.1% (95%; CI: 0.6-2.1) were confirmed to be HBsAg-positive. Of those samples tested for anti-HBs (n = 1033), only 74.4% presented a serological profile analogous to that elicited by hepatitis B vaccination. Among HBsAg-positive samples (n = 29), 72.4% were HBV DNA-positive, and from these, 18 were sequenced. HBV genotypes A, F, and G were found in 55.5%, 38.9%, and 5.6%, respectively. This study indicates high prevalence rates of MSM HBV exposure and a low positivity index for the serological marker of HBV vaccine immunity. These findings may contribute to the discussion of strategies to prevent hepatitis B and reinforce the importance of promoting HBV vaccination in this key population.