A total of 775 serum samples from men and women working in hospitals in Dakar, Senegal, were tested for serological markers of hepatitis B virus (HBV). HBsAg was detected in 17.8% of the subjects, and 79.2% of the subjects were anti-HBc positive. Among HBsAg carriers 0.04% (5) subjects were HBeAg positive, 0.03% (4) were HBV-DNA positive, and 5.8% (8) were also anti-Delta positive. HBsAg seropositivity was independent of sex and inversely related to age. Duration of service in the hospital was an important predictor of HBsAg seropositivity and the prevalence of seropositive subjects peaked between 2 and 3 years of employment (OR = 1.9; 95% confidence interval [CI] = 1.1–3.3, when compared to subjects who worked 1 year of less). This peak was critical in the Department of Dentistry, where subjects who worked for 2–3 years experienced a fourfold increase in the risk of HBV infection (OR =4.0; 95% CI = 1.8–9.0). Adjusting for age and sex did not modify the results. Within the Department of Dentistry, 15 subjects were HBsAg positive but anti-HBc negative; 12 subjects were retested 1 year later and did not present any markers of past or current HBV infection. These results confirm the increased risk of HBV infection among hospital workers and suggest the presence of HBV variant(s) in Senegal.