In France, several successive changes in anti-hepatitis B virus (HBV) vaccination policies occurred since 1982. We estimated the incidence and prevalence of HBV infection according to years of birth 1960 to 1994 in a large sentinel cohort to evaluate the epidemiology of HBV during vaccination policy changes. A retrospective cohort study included data from all HIV, HBV and hepatitis C virus (HCV) screening facilities in Grand Lyon. From 2005 to 2010, all 57113 individuals with complete HBV serologic status were enrolled. Survival analyses modeled separately various ages in each birth cohort. The proportion of immunized individuals increased in birth cohorts 1978 to 1984 (up to 58.3% (95% confidence interval (CI), 43.3-68.2 at age 15). In post-1985 birth cohorts, this proportion decreased to 19.5% (95% CI, 15.5-24) in birth cohort 1987 at age 15. Probability of past or current HBV infection increased constantly in birth cohorts 1960 to 1967, up to 12% (95% CI, 10.4-14) at age 30, then decreased gradually in birth cohorts 1968 to 1991, down to 0.9% (95% CI, 0.7-1.2) at age 17.5. In post-1991 cohorts, the probability of HBV infection increased again, up to 2.5% (95% CI, 1.7-3.6) at age 17.5. HBV incidence fluctuated between 5 and 8 per 1000 person-years in pre-1986 birth cohorts, decreased to 2.1 (95% CI, 1.5-2.7) in birth cohorts 1986 to 1991 but rebounded to 5 (95% CI, 3.5-7.1) in post-1991 birth cohorts. HBV incidence was remarkably high in young adults with noticeable variations concomitantly to vaccination policy changes. A dramatic decline in immunization rate was temporally associated with a sharp rebound of infection after withdrawal of systematic adolescent vaccination in 1998.