To explore changes in molecular epidemiology of acute viral hepatitis B (AVH-B), hepatitis B virus (HBV) genotypes were determined by direct sequencing of the Pre-S-S region in 123 consecutive patients, with AVH-B observed in Naples or its surroundings in the last decade (group AVH-B) and in 123 HBV chronic carriers [chronic carrier of HBV (CC-B) group] from the same areas, who had been hepatitis B surface antigen-positive for more than 10 years. Genotype D was less frequently detected in patients with AVH-B than in those in the CC-B group (76.4%vs 97.5%, P < 0.0001). In the AVH-B group, intravenous drug addiction (IVDA) was the prevalent risk factor (55.3%) for acquiring HBV in the 94 patients with HBV genotype D, but it was rarely recorded (6.9%) in the 29 patients with genotypes non-D (P < 0.0001); unsafe sexual intercourse was prevalent in patients with genotype non-D (72.3%) and less frequent in those with genotype D (28.8%, P < 0.005). In the AVH-B group, the prevalence of non-D genotypes increased during the observation period from 11.1% in 1999-2003 to 41.1% in 2004-2008 (P < 0.0005), paralleling the increase in the prevalence of patients with unsafe sexual intercourse; similarly, the progressive decrease in IVDA paralleled the decrease in the prevalence of genotype D (from 88.3% in 1999-2003 to 11.7% in 2004-2008). The prevalence of HBV non-D genotypes recorded in the last 10 years in AVH-B in this area shows a progressive increase, most probably because of recent changes in HBV epidemiology, namely, the HBV mass vaccination campaign and increased immigration from areas with high HBV endemicity.