Background: Prison populations are at high risk for hepatitis B virus (HBV) infection. The aim of this study was to assess the prevalence, incidence, HBV associated factors and circulating genotypes/subtypes. Methods: A total of 3,368 prisoners from 12 closed prisons were randomly recruited for a cross-sectional study. In addition, a cohort study was conducted 12 months later and included 1,656 individuals. Participants underwent an interview and blood collection for the detection of HBV serological markers and HBV-DNA phylogenetic analysis. Results: HBV exposure (anti-HBc + ) was 9.8% (95% CI: 8.8-10.8); 11.2% were female and 9.6% were male. HBsAg+ was 0.6%. Only 31.4% of the participants had HBV vaccination-like profile (anti-HBs+ alone; 30.4% male vs. 36.8% female; p = 0.004). Most individuals were susceptible to HBV (60.2% female vs. 52.2% male, p = 0.001). HBV isolates were classified as genotypes A (45.4%), D (27.3%) and F (27.3%). In males, HBV exposure was associated with increased age. Male prisoners had more evidence of HCV/HBV co-infection (10.7%) than females (3.4%) and the frequency of Treponema pallidum infection among prisoners who had been exposed to HBV was higher in female prisoners when compared with male (39.7% vs.19.1%). The incidence of HBV was 0.18/100 person-years (95% CI: 0.12%–0.25%). Conclusions: Our results indicate a high prevalence of HBV exposure in prisoners. Despite the low incidence of this infection, the occurrence of new cases indicates the need to implement preventive measures.